Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan
Identifieur interne : 000F98 ( Ncbi/Merge ); précédent : 000F97; suivant : 000F99Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan
Auteurs : Sumiko Ikari [Japon] ; Shiou-Ling Lu [Japon] ; Feike Hao [Japon, République populaire de Chine] ; Kenta Imai [Japon] ; Yasuhiro Araki [Japon] ; Yo-Hei Yamamoto [Japon] ; Chao-Yuan Tsai [Japon] ; Yumi Nishiyama [Japon] ; Nobukazu Shitan [Japon] ; Tamotsu Yoshimori [Japon] ; Takanobu Otomo [Japon] ; Takeshi Noda [Japon]Source :
- PLoS ONE [ 1932-6203 ] ; 2020.
Abstract
Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.
Url:
DOI: 10.1371/journal.pone.0230156
PubMed: 32134989
PubMed Central: 7058311
Links toward previous steps (curation, corpus...)
- to stream Pmc, to step Corpus: 000783
- to stream Pmc, to step Curation: 000783
- to stream Pmc, to step Checkpoint: 000040
Links to Exploration step
PMC:7058311Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan</title><author><name sortKey="Ikari, Sumiko" sort="Ikari, Sumiko" uniqKey="Ikari S" first="Sumiko" last="Ikari">Sumiko Ikari</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Lu, Shiou Ling" sort="Lu, Shiou Ling" uniqKey="Lu S" first="Shiou-Ling" last="Lu">Shiou-Ling Lu</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Hao, Feike" sort="Hao, Feike" uniqKey="Hao F" first="Feike" last="Hao">Feike Hao</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation><affiliation wicri:level="3"><nlm:aff id="aff003"><addr-line>China National Research Institute of Food and Fermentation Industries, Beijing, China</addr-line></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>China National Research Institute of Food and Fermentation Industries, Beijing</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Imai, Kenta" sort="Imai, Kenta" uniqKey="Imai K" first="Kenta" last="Imai">Kenta Imai</name><affiliation wicri:level="1"><nlm:aff id="aff004"><addr-line>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Araki, Yasuhiro" sort="Araki, Yasuhiro" uniqKey="Araki Y" first="Yasuhiro" last="Araki">Yasuhiro Araki</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Yamamoto, Yo Hei" sort="Yamamoto, Yo Hei" uniqKey="Yamamoto Y" first="Yo-Hei" last="Yamamoto">Yo-Hei Yamamoto</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Tsai, Chao Yuan" sort="Tsai, Chao Yuan" uniqKey="Tsai C" first="Chao-Yuan" last="Tsai">Chao-Yuan Tsai</name><affiliation wicri:level="1"><nlm:aff id="aff005"><addr-line>Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Nishiyama, Yumi" sort="Nishiyama, Yumi" uniqKey="Nishiyama Y" first="Yumi" last="Nishiyama">Yumi Nishiyama</name><affiliation wicri:level="1"><nlm:aff id="aff006"><addr-line>Medicinal Botanical Garden, Kobe Pharmaceutical University, Kobe, Hyogo, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Medicinal Botanical Garden, Kobe Pharmaceutical University, Kobe, Hyogo</wicri:regionArea><wicri:noRegion>Hyogo</wicri:noRegion></affiliation></author><author><name sortKey="Shitan, Nobukazu" sort="Shitan, Nobukazu" uniqKey="Shitan N" first="Nobukazu" last="Shitan">Nobukazu Shitan</name><affiliation wicri:level="1"><nlm:aff id="aff007"><addr-line>Laboratory of Medicinal Cell Biology, Kobe Pharmaceutical University, Kobe, Hyogo, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Laboratory of Medicinal Cell Biology, Kobe Pharmaceutical University, Kobe, Hyogo</wicri:regionArea><wicri:noRegion>Hyogo</wicri:noRegion></affiliation></author><author><name sortKey="Yoshimori, Tamotsu" sort="Yoshimori, Tamotsu" uniqKey="Yoshimori T" first="Tamotsu" last="Yoshimori">Tamotsu Yoshimori</name><affiliation wicri:level="1"><nlm:aff id="aff004"><addr-line>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Otomo, Takanobu" sort="Otomo, Takanobu" uniqKey="Otomo T" first="Takanobu" last="Otomo">Takanobu Otomo</name><affiliation wicri:level="1"><nlm:aff id="aff008"><addr-line>Department of Molecular and Genetic Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Molecular and Genetic Medicine, Kawasaki Medical School, Kurashiki, Okayama</wicri:regionArea><wicri:noRegion>Okayama</wicri:noRegion></affiliation></author><author><name sortKey="Noda, Takeshi" sort="Noda, Takeshi" uniqKey="Noda T" first="Takeshi" last="Noda">Takeshi Noda</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32134989</idno><idno type="pmc">7058311</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058311</idno><idno type="RBID">PMC:7058311</idno><idno type="doi">10.1371/journal.pone.0230156</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">000783</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000783</idno><idno type="wicri:Area/Pmc/Curation">000783</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000783</idno><idno type="wicri:Area/Pmc/Checkpoint">000040</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000040</idno><idno type="wicri:Area/Ncbi/Merge">000F98</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan</title><author><name sortKey="Ikari, Sumiko" sort="Ikari, Sumiko" uniqKey="Ikari S" first="Sumiko" last="Ikari">Sumiko Ikari</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Lu, Shiou Ling" sort="Lu, Shiou Ling" uniqKey="Lu S" first="Shiou-Ling" last="Lu">Shiou-Ling Lu</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Hao, Feike" sort="Hao, Feike" uniqKey="Hao F" first="Feike" last="Hao">Feike Hao</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation><affiliation wicri:level="3"><nlm:aff id="aff003"><addr-line>China National Research Institute of Food and Fermentation Industries, Beijing, China</addr-line></nlm:aff><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>China National Research Institute of Food and Fermentation Industries, Beijing</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Imai, Kenta" sort="Imai, Kenta" uniqKey="Imai K" first="Kenta" last="Imai">Kenta Imai</name><affiliation wicri:level="1"><nlm:aff id="aff004"><addr-line>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Araki, Yasuhiro" sort="Araki, Yasuhiro" uniqKey="Araki Y" first="Yasuhiro" last="Araki">Yasuhiro Araki</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Yamamoto, Yo Hei" sort="Yamamoto, Yo Hei" uniqKey="Yamamoto Y" first="Yo-Hei" last="Yamamoto">Yo-Hei Yamamoto</name><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Tsai, Chao Yuan" sort="Tsai, Chao Yuan" uniqKey="Tsai C" first="Chao-Yuan" last="Tsai">Chao-Yuan Tsai</name><affiliation wicri:level="1"><nlm:aff id="aff005"><addr-line>Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Nishiyama, Yumi" sort="Nishiyama, Yumi" uniqKey="Nishiyama Y" first="Yumi" last="Nishiyama">Yumi Nishiyama</name><affiliation wicri:level="1"><nlm:aff id="aff006"><addr-line>Medicinal Botanical Garden, Kobe Pharmaceutical University, Kobe, Hyogo, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Medicinal Botanical Garden, Kobe Pharmaceutical University, Kobe, Hyogo</wicri:regionArea><wicri:noRegion>Hyogo</wicri:noRegion></affiliation></author><author><name sortKey="Shitan, Nobukazu" sort="Shitan, Nobukazu" uniqKey="Shitan N" first="Nobukazu" last="Shitan">Nobukazu Shitan</name><affiliation wicri:level="1"><nlm:aff id="aff007"><addr-line>Laboratory of Medicinal Cell Biology, Kobe Pharmaceutical University, Kobe, Hyogo, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Laboratory of Medicinal Cell Biology, Kobe Pharmaceutical University, Kobe, Hyogo</wicri:regionArea><wicri:noRegion>Hyogo</wicri:noRegion></affiliation></author><author><name sortKey="Yoshimori, Tamotsu" sort="Yoshimori, Tamotsu" uniqKey="Yoshimori T" first="Tamotsu" last="Yoshimori">Tamotsu Yoshimori</name><affiliation wicri:level="1"><nlm:aff id="aff004"><addr-line>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author><author><name sortKey="Otomo, Takanobu" sort="Otomo, Takanobu" uniqKey="Otomo T" first="Takanobu" last="Otomo">Takanobu Otomo</name><affiliation wicri:level="1"><nlm:aff id="aff008"><addr-line>Department of Molecular and Genetic Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Molecular and Genetic Medicine, Kawasaki Medical School, Kurashiki, Okayama</wicri:regionArea><wicri:noRegion>Okayama</wicri:noRegion></affiliation></author><author><name sortKey="Noda, Takeshi" sort="Noda, Takeshi" uniqKey="Noda T" first="Takeshi" last="Noda">Takeshi Noda</name><affiliation wicri:level="1"><nlm:aff id="aff001"><addr-line>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="aff002"><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></nlm:aff><country xml:lang="fr">Japon</country><wicri:regionArea>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka</wicri:regionArea><wicri:noRegion>Osaka</wicri:noRegion></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca<sup>2+</sup> dependent manner. Cytosolic Ca<sup>2+</sup> concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca<sup>2+</sup> increase, thereby suppressing induction of autophagy.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Mizushima, N" uniqKey="Mizushima N">N. Mizushima</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, Yg" uniqKey="Zhao Y">YG Zhao</name></author><author><name sortKey="Zhang, H" uniqKey="Zhang H">H Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Luo, T" uniqKey="Luo T">T Luo</name></author><author><name sortKey="Fu, J" uniqKey="Fu J">J Fu</name></author><author><name sortKey="Xu, A" uniqKey="Xu A">A Xu</name></author><author><name sortKey="Su, B" uniqKey="Su B">B Su</name></author><author><name sortKey="Ren, Y" uniqKey="Ren Y">Y Ren</name></author><author><name sortKey="Li, N" uniqKey="Li N">N Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, M" uniqKey="Liu M">M Liu</name></author><author><name sortKey="Jiang, L" uniqKey="Jiang L">L Jiang</name></author><author><name sortKey="Fu, X" uniqKey="Fu X">X Fu</name></author><author><name sortKey="Wang, W" uniqKey="Wang W">W Wang</name></author><author><name sortKey="Ma, J" uniqKey="Ma J">J Ma</name></author><author><name sortKey="Tian, T" uniqKey="Tian T">T Tian</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kinsey, Cg" uniqKey="Kinsey C">CG Kinsey</name></author><author><name sortKey="Camolotto, Sa" uniqKey="Camolotto S">SA Camolotto</name></author><author><name sortKey="Boespflug, Am" uniqKey="Boespflug A">AM Boespflug</name></author><author><name sortKey="Guillen, Kp" uniqKey="Guillen K">KP Guillen</name></author><author><name sortKey="Foth, M" uniqKey="Foth M">M Foth</name></author><author><name sortKey="Truong, A" uniqKey="Truong A">A Truong</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yusuf, Ih" uniqKey="Yusuf I">IH Yusuf</name></author><author><name sortKey="Sharma, S" uniqKey="Sharma S">S Sharma</name></author><author><name sortKey="Luqmani, R" uniqKey="Luqmani R">R Luqmani</name></author><author><name sortKey="Downes, Sm" uniqKey="Downes S">SM Downes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kroemer, Guido" uniqKey="Kroemer G">Guido Kroemer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rubinsztein, Dc" uniqKey="Rubinsztein D">DC Rubinsztein</name></author><author><name sortKey="Codogno, P" uniqKey="Codogno P">P Codogno</name></author><author><name sortKey="Levine, B" uniqKey="Levine B">B Levine</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, Sf" uniqKey="Wang S">SF Wang</name></author><author><name sortKey="Wu, My" uniqKey="Wu M">MY Wu</name></author><author><name sortKey="Cai, Cz" uniqKey="Cai C">CZ Cai</name></author><author><name sortKey="Li, M" uniqKey="Li M">M Li</name></author><author><name sortKey="Lu, Jh" uniqKey="Lu J">JH Lu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yu, F" uniqKey="Yu F">F Yu</name></author><author><name sortKey="Takahashi, T" uniqKey="Takahashi T">T Takahashi</name></author><author><name sortKey="Moriya, J" uniqKey="Moriya J">J Moriya</name></author><author><name sortKey="Kawaura, K" uniqKey="Kawaura K">K Kawaura</name></author><author><name sortKey="Yamakawa, J" uniqKey="Yamakawa J">J Yamakawa</name></author><author><name sortKey="Kusaka, K" uniqKey="Kusaka K">K Kusaka</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Takanashi, K" uniqKey="Takanashi K">K Takanashi</name></author><author><name sortKey="Dan, K" uniqKey="Dan K">K Dan</name></author><author><name sortKey="Kanzaki, S" uniqKey="Kanzaki S">S Kanzaki</name></author><author><name sortKey="Hasegawa, H" uniqKey="Hasegawa H">H Hasegawa</name></author><author><name sortKey="Watanabe, K" uniqKey="Watanabe K">K Watanabe</name></author><author><name sortKey="Ogawa, K" uniqKey="Ogawa K">K Ogawa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Okubo, S" uniqKey="Okubo S">S Okubo</name></author><author><name sortKey="Komori, H" uniqKey="Komori H">H Komori</name></author><author><name sortKey="Kuwahara, A" uniqKey="Kuwahara A">A Kuwahara</name></author><author><name sortKey="Ohta, T" uniqKey="Ohta T">T Ohta</name></author><author><name sortKey="Shoyama, Y" uniqKey="Shoyama Y">Y Shoyama</name></author><author><name sortKey="Uto, T" uniqKey="Uto T">T Uto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kimura, S" uniqKey="Kimura S">S Kimura</name></author><author><name sortKey="Fujita, N" uniqKey="Fujita N">N Fujita</name></author><author><name sortKey="Noda, T" uniqKey="Noda T">T Noda</name></author><author><name sortKey="Yoshimori, T" uniqKey="Yoshimori T">T Yoshimori</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chang, C" uniqKey="Chang C">C Chang</name></author><author><name sortKey="Young, Ln" uniqKey="Young L">LN Young</name></author><author><name sortKey="Morris, Kl" uniqKey="Morris K">KL Morris</name></author><author><name sortKey="Von Bulow, S" uniqKey="Von Bulow S">S von Bülow</name></author><author><name sortKey="Schoneberg, J" uniqKey="Schoneberg J">J Schöneberg</name></author><author><name sortKey="Yamamoto Imoto, H" uniqKey="Yamamoto Imoto H">H Yamamoto-Imoto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kageyama, S" uniqKey="Kageyama S">S Kageyama</name></author><author><name sortKey="Omori, H" uniqKey="Omori H">H Omori</name></author><author><name sortKey="Saitoh, T" uniqKey="Saitoh T">T Saitoh</name></author><author><name sortKey="Sone, T" uniqKey="Sone T">T Sone</name></author><author><name sortKey="Guan, Jl" uniqKey="Guan J">JL Guan</name></author><author><name sortKey="Akira, S" uniqKey="Akira S">S Akira</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hao, F" uniqKey="Hao F">F Hao</name></author><author><name sortKey="Kondo, K" uniqKey="Kondo K">K Kondo</name></author><author><name sortKey="Itoh, T" uniqKey="Itoh T">T Itoh</name></author><author><name sortKey="Ikari, S" uniqKey="Ikari S">S Ikari</name></author><author><name sortKey="Nada, S" uniqKey="Nada S">S Nada</name></author><author><name sortKey="Okada, M" uniqKey="Okada M">M Okada</name></author><author><name sortKey="Noda, T" uniqKey="Noda T">T Noda</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Saitoh, T" uniqKey="Saitoh T">T Saitoh</name></author><author><name sortKey="Nakayama, M" uniqKey="Nakayama M">M Nakayama</name></author><author><name sortKey="Nakano, H" uniqKey="Nakano H">H Nakano</name></author><author><name sortKey="Yagita, H" uniqKey="Yagita H">H Yagita</name></author><author><name sortKey="Yamamoto, N" uniqKey="Yamamoto N">N Yamamoto</name></author><author><name sortKey="Yamaoka, S" uniqKey="Yamaoka S">S Yamaoka</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bloor, S" uniqKey="Bloor S">S Bloor</name></author><author><name sortKey="Maelfait, J" uniqKey="Maelfait J">J Maelfait</name></author><author><name sortKey="Krumbach, R" uniqKey="Krumbach R">R Krumbach</name></author><author><name sortKey="Beyaert, R" uniqKey="Beyaert R">R Beyaert</name></author><author><name sortKey="Randow, F" uniqKey="Randow F">F Randow</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sardiello, Marco" uniqKey="Sardiello M">Marco Sardiello</name></author><author><name sortKey="Michela, Palimieri" uniqKey="Michela P">Palimieri Michela</name></author><author><name sortKey="Di Ronza Alberto, Medina Diego Luis Vm" uniqKey="Di Ronza Alberto M">Medina Diego Luis VM Di Ronza Alberto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nakai, J" uniqKey="Nakai J">J Nakai</name></author><author><name sortKey="Ohkura, M" uniqKey="Ohkura M">M Ohkura</name></author><author><name sortKey="Imoto, K" uniqKey="Imoto K">K Imoto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kimura, S" uniqKey="Kimura S">S Kimura</name></author><author><name sortKey="Noda, T" uniqKey="Noda T">T Noda</name></author><author><name sortKey="Yoshimori, T" uniqKey="Yoshimori T">T Yoshimori</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thoreen, Cc" uniqKey="Thoreen C">CC Thoreen</name></author><author><name sortKey="Kang, Sa" uniqKey="Kang S">SA Kang</name></author><author><name sortKey="Chang, Jw" uniqKey="Chang J">JW Chang</name></author><author><name sortKey="Liu, Q" uniqKey="Liu Q">Q Liu</name></author><author><name sortKey="Zhang, J" uniqKey="Zhang J">J Zhang</name></author><author><name sortKey="Gao, Y" uniqKey="Gao Y">Y Gao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yamamoto, A" uniqKey="Yamamoto A">A Yamamoto</name></author><author><name sortKey="Tagawa, Y" uniqKey="Tagawa Y">Y Tagawa</name></author><author><name sortKey="Yoshimori, T" uniqKey="Yoshimori T">T Yoshimori</name></author><author><name sortKey="Moriyama, Y" uniqKey="Moriyama Y">Y Moriyama</name></author><author><name sortKey="Masaki, R" uniqKey="Masaki R">R Masaki</name></author><author><name sortKey="Tashiro, Y" uniqKey="Tashiro Y">Y Tashiro</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lamark, T" uniqKey="Lamark T">T Lamark</name></author><author><name sortKey="Johansen, T" uniqKey="Johansen T">T Johansen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lippai, M" uniqKey="Lippai M">M Lippai</name></author><author><name sortKey="Low, P" uniqKey="Low P">P Low</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tanida, I" uniqKey="Tanida I">I Tanida</name></author><author><name sortKey="Miematsu Ikeguchi, N" uniqKey="Miematsu Ikeguchi N">N Miematsu-ikeguchi</name></author><author><name sortKey="Ueno, T" uniqKey="Ueno T">T Ueno</name></author><author><name sortKey="Kominami, E" uniqKey="Kominami E">E Kominami</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mizushima, N" uniqKey="Mizushima N">N Mizushima</name></author><author><name sortKey="Yamamoto, A" uniqKey="Yamamoto A">A Yamamoto</name></author><author><name sortKey="Hatano, M" uniqKey="Hatano M">M Hatano</name></author><author><name sortKey="Kobayashi, Y" uniqKey="Kobayashi Y">Y Kobayashi</name></author><author><name sortKey="Kabey, Y" uniqKey="Kabey Y">Y Kabey</name></author><author><name sortKey="Suzuki, K" uniqKey="Suzuki K">K Suzuki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hara, T" uniqKey="Hara T">T Hara</name></author><author><name sortKey="Takamura, A" uniqKey="Takamura A">A Takamura</name></author><author><name sortKey="Kishi, C" uniqKey="Kishi C">C Kishi</name></author><author><name sortKey="Iemura, S" uniqKey="Iemura S">S Iemura</name></author><author><name sortKey="Natsume, T" uniqKey="Natsume T">T Natsume</name></author><author><name sortKey="Guan J, L" uniqKey="Guan J L">L Guan J</name></author><author><name sortKey="Mizushima, N" uniqKey="Mizushima N">N Mizushima</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Proikas Cezanne, T" uniqKey="Proikas Cezanne T">T Proikas-Cezanne</name></author><author><name sortKey="Takacs, Z" uniqKey="Takacs Z">Z Takacs</name></author><author><name sortKey="Donnes, P" uniqKey="Donnes P">P Dönnes</name></author><author><name sortKey="Kohlbacher, O" uniqKey="Kohlbacher O">O Kohlbacher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Noda, T" uniqKey="Noda T">T. Noda</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Settembre, C" uniqKey="Settembre C">C Settembre</name></author><author><name sortKey="Polito, Va" uniqKey="Polito V">VA Polito</name></author><author><name sortKey="Arencibia, Mg" uniqKey="Arencibia M">MG Arencibia</name></author><author><name sortKey="Vetrini, F" uniqKey="Vetrini F">F Vetrini</name></author><author><name sortKey="Erdin, S" uniqKey="Erdin S">S Erdin</name></author><author><name sortKey="Erdin, Su" uniqKey="Erdin S">SU Erdin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mizushima, N" uniqKey="Mizushima N">N. Mizushima</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fingar, Dc" uniqKey="Fingar D">DC Fingar</name></author><author><name sortKey="Richardson, Cj" uniqKey="Richardson C">CJ Richardson</name></author><author><name sortKey="Tee, Ar" uniqKey="Tee A">AR Tee</name></author><author><name sortKey="Cheatham, L" uniqKey="Cheatham L">L Cheatham</name></author><author><name sortKey="Tsou, C" uniqKey="Tsou C">C Tsou</name></author><author><name sortKey="Blenis, J" uniqKey="Blenis J">J Blenis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zatloukal, B" uniqKey="Zatloukal B">B Zatloukal</name></author><author><name sortKey="Kufferath, I" uniqKey="Kufferath I">I Kufferath</name></author><author><name sortKey="Thueringer, A" uniqKey="Thueringer A">A Thueringer</name></author><author><name sortKey="Landegren, U" uniqKey="Landegren U">U Landegren</name></author><author><name sortKey="Zatloukal, K" uniqKey="Zatloukal K">K Zatloukal</name></author><author><name sortKey="Haybaeck, J" uniqKey="Haybaeck J">J Haybaeck</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hosokawa, N" uniqKey="Hosokawa N">N Hosokawa</name></author><author><name sortKey="Hara, T" uniqKey="Hara T">T Hara</name></author><author><name sortKey="Takeshi Kaizuka, Ck" uniqKey="Takeshi Kaizuka C">CK Takeshi Kaizuka</name></author><author><name sortKey="Akito, Takamura" uniqKey="Akito T">Takamura Akito</name></author><author><name sortKey="Yutaka Miura, Si" uniqKey="Yutaka Miura S">SI Yutaka Miura</name></author><author><name sortKey="Natsume, T" uniqKey="Natsume T">T Natsume</name></author><author><name sortKey="Kenji Takehana, Ny" uniqKey="Kenji Takehana N">NY Kenji Takehana</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Martina, Ja" uniqKey="Martina J">JA Martina</name></author><author><name sortKey="Puertollano, R" uniqKey="Puertollano R">R Puertollano</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schalm, Ss" uniqKey="Schalm S">SS Schalm</name></author><author><name sortKey="Blenis, J" uniqKey="Blenis J">J Blenis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Medina, Dl" uniqKey="Medina D">DL Medina</name></author><author><name sortKey="Di Paola, S" uniqKey="Di Paola S">S Di Paola</name></author><author><name sortKey="Peluso, I" uniqKey="Peluso I">I Peluso</name></author><author><name sortKey="Armani, A" uniqKey="Armani A">A Armani</name></author><author><name sortKey="De Stefani, D" uniqKey="De Stefani D">D De Stefani</name></author><author><name sortKey="Venditti, R" uniqKey="Venditti R">R Venditti</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mckeon, F" uniqKey="Mckeon F">F. McKeon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Aramburu, J" uniqKey="Aramburu J">J Aramburu</name></author><author><name sortKey="Yaffe, Mb" uniqKey="Yaffe M">MB Yaffe</name></author><author><name sortKey="L Pez Rodriguez, C" uniqKey="L Pez Rodriguez C">C López-Rodríguez</name></author><author><name sortKey="Cantley, Lc" uniqKey="Cantley L">LC Cantley</name></author><author><name sortKey="Hogan, Pg" uniqKey="Hogan P">PG Hogan</name></author><author><name sortKey="Rao, A" uniqKey="Rao A">A Rao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hagen, Bm" uniqKey="Hagen B">BM Hagen</name></author><author><name sortKey="Boyman, L" uniqKey="Boyman L">L Boyman</name></author><author><name sortKey="Kao, Jpy" uniqKey="Kao J">JPY Kao</name></author><author><name sortKey="Lederer, Wj" uniqKey="Lederer W">WJ Lederer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="H Yer Hansen, M" uniqKey="H Yer Hansen M">M Høyer-Hansen</name></author><author><name sortKey="Bastholm, L" uniqKey="Bastholm L">L Bastholm</name></author><author><name sortKey="Szyniarowski, P" uniqKey="Szyniarowski P">P Szyniarowski</name></author><author><name sortKey="Campanella, M" uniqKey="Campanella M">M Campanella</name></author><author><name sortKey="Szabadkai, G" uniqKey="Szabadkai G">G Szabadkai</name></author><author><name sortKey="Farkas, T" uniqKey="Farkas T">T Farkas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stewart, Aa" uniqKey="Stewart A">AA Stewart</name></author><author><name sortKey="Ingebritsen, Ts" uniqKey="Ingebritsen T">TS Ingebritsen</name></author><author><name sortKey="Manalan, A" uniqKey="Manalan A">A Manalan</name></author><author><name sortKey="Klee, Cb" uniqKey="Klee C">CB Klee</name></author><author><name sortKey="Cohen, P" uniqKey="Cohen P">P Cohen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Oka, T" uniqKey="Oka T">T Oka</name></author><author><name sortKey="Sato, K" uniqKey="Sato K">K Sato</name></author><author><name sortKey="Hori, M" uniqKey="Hori M">M Hori</name></author><author><name sortKey="Ozaki, H" uniqKey="Ozaki H">H Ozaki</name></author><author><name sortKey="Karaki, H" uniqKey="Karaki H">H Karaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yoshikawa, T" uniqKey="Yoshikawa T">T. Yoshikawa</name></author><author><name sortKey="Takahashi, S" uniqKey="Takahashi S">S Takahashi</name></author><author><name sortKey="Ichikawa, H" uniqKey="Ichikawa H">H Ichikawa</name></author><author><name sortKey="Takano, H" uniqKey="Takano H">H Takano</name></author><author><name sortKey="Tasaki, N" uniqKey="Tasaki N">N Tasaki</name></author><author><name sortKey="Yasuda, M" uniqKey="Yasuda M">M Yasuda</name></author><author><name sortKey="Nito, Y" uniqKey="Nito Y">Y Nito</name></author><author><name sortKey="Tanigaki, T" uniqKey="Tanigaki T">T Tanigaki</name></author><author><name sortKey="Kondo, M" uniqKey="Kondo M">M Kondo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ohta, Y" uniqKey="Ohta Y">Y Ohta</name></author><author><name sortKey="Kobayashi, T" uniqKey="Kobayashi T">T Kobayashi</name></author><author><name sortKey="Hayashi, T" uniqKey="Hayashi T">T Hayashi</name></author><author><name sortKey="Inui, K" uniqKey="Inui K">K Inui</name></author><author><name sortKey="Yoshino, J" uniqKey="Yoshino J">J Yoshino</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hashimoto, D" uniqKey="Hashimoto D">D Hashimoto</name></author><author><name sortKey="Ohmuraya, M" uniqKey="Ohmuraya M">M Ohmuraya</name></author><author><name sortKey="Hirota, M" uniqKey="Hirota M">M Hirota</name></author><author><name sortKey="Yamamoto, A" uniqKey="Yamamoto A">A Yamamoto</name></author><author><name sortKey="Suyama, K" uniqKey="Suyama K">K Suyama</name></author><author><name sortKey="Ida, S" uniqKey="Ida S">S Ida</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, Ey" uniqKey="Chan E">EY Chan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pen, S" uniqKey="Pen S">S Pen</name></author><author><name sortKey="Vega Rubin De Celis, S" uniqKey="Vega Rubin De Celis S">S Vega-rubin-de-celis</name></author><author><name sortKey="Schwartz, Jc" uniqKey="Schwartz J">JC Schwartz</name></author><author><name sortKey="Wolff, Nc" uniqKey="Wolff N">NC Wolff</name></author><author><name sortKey="Tran, Tat" uniqKey="Tran T">TAT Tran</name></author><author><name sortKey="Zou, L" uniqKey="Zou L">L Zou</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Decuypere, Jp" uniqKey="Decuypere J">JP Decuypere</name></author><author><name sortKey="Bultynck, G" uniqKey="Bultynck G">G Bultynck</name></author><author><name sortKey="Parys, Jb" uniqKey="Parys J">JB Parys</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Decuypere, Jp" uniqKey="Decuypere J">JP Decuypere</name></author><author><name sortKey="Welkenhuyzen, K" uniqKey="Welkenhuyzen K">K Welkenhuyzen</name></author><author><name sortKey="Luyten, T" uniqKey="Luyten T">T Luyten</name></author><author><name sortKey="Ponsaerts, R" uniqKey="Ponsaerts R">R Ponsaerts</name></author><author><name sortKey="Dewaele, M" uniqKey="Dewaele M">M Dewaele</name></author><author><name sortKey="Molg, J" uniqKey="Molg J">J Molgó</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, CA USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32134989</article-id><article-id pub-id-type="pmc">7058311</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0230156</article-id><article-id pub-id-type="publisher-id">PONE-D-19-29517</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Cell Biology</subject><subj-group><subject>Cell Processes</subject><subj-group><subject>Cell Death</subject><subj-group><subject>Autophagic Cell Death</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject><subj-group><subject>Biological cultures</subject><subj-group><subject>Cell lines</subject><subj-group><subject>HeLa cells</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject><subj-group><subject>Biological cultures</subject><subj-group><subject>Cell cultures</subject><subj-group><subject>Cultured tumor cells</subject><subj-group><subject>HeLa cells</subject></subj-group></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Medicine and Health Sciences</subject><subj-group><subject>Complementary and Alternative Medicine</subject><subj-group><subject>Traditional Medicine</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and Analysis Methods</subject><subj-group><subject>Cell Enumeration Techniques</subject></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Cell Biology</subject><subj-group><subject>Cellular Structures and Organelles</subject><subj-group><subject>Lysosomes</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and Analysis Methods</subject><subj-group><subject>Imaging Techniques</subject><subj-group><subject>Fluorescence Imaging</subject></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject><subj-group><subject>Cell Biology</subject><subj-group><subject>Signal Transduction</subject><subj-group><subject>Cell Signaling</subject></subj-group></subj-group></subj-group></subj-group><subj-group subj-group-type="Discipline-v3"><subject>Research and Analysis Methods</subject><subj-group><subject>Microscopy</subject><subj-group><subject>Light Microscopy</subject><subj-group><subject>Fluorescence Microscopy</subject></subj-group></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan</article-title><alt-title alt-title-type="running-head">Autophagy via calcium-dependent TFEB axis is suppressed by Shigyakusan</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Ikari</surname><given-names>Sumiko</given-names></name><role content-type="http://credit.casrai.org/">Conceptualization</role><role content-type="http://credit.casrai.org/">Data curation</role><role content-type="http://credit.casrai.org/">Formal analysis</role><role content-type="http://credit.casrai.org/">Investigation</role><role content-type="http://credit.casrai.org/">Writing – original draft</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lu</surname><given-names>Shiou-Ling</given-names></name><role content-type="http://credit.casrai.org/">Investigation</role><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hao</surname><given-names>Feike</given-names></name><role content-type="http://credit.casrai.org/">Methodology</role><xref ref-type="aff" rid="aff002"><sup>2</sup></xref><xref ref-type="aff" rid="aff003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Imai</surname><given-names>Kenta</given-names></name><role content-type="http://credit.casrai.org/">Supervision</role><xref ref-type="aff" rid="aff004"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Araki</surname><given-names>Yasuhiro</given-names></name><role content-type="http://credit.casrai.org/">Methodology</role><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Yamamoto</surname><given-names>Yo-hei</given-names></name><role content-type="http://credit.casrai.org/">Methodology</role><xref ref-type="aff" rid="aff002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Tsai</surname><given-names>Chao-Yuan</given-names></name><role content-type="http://credit.casrai.org/">Resources</role><xref ref-type="aff" rid="aff005"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Nishiyama</surname><given-names>Yumi</given-names></name><role content-type="http://credit.casrai.org/">Investigation</role><xref ref-type="aff" rid="aff006"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Shitan</surname><given-names>Nobukazu</given-names></name><role content-type="http://credit.casrai.org/">Conceptualization</role><role content-type="http://credit.casrai.org/">Methodology</role><xref ref-type="aff" rid="aff007"><sup>7</sup></xref></contrib><contrib contrib-type="author"><name><surname>Yoshimori</surname><given-names>Tamotsu</given-names></name><role content-type="http://credit.casrai.org/">Conceptualization</role><xref ref-type="aff" rid="aff004"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Otomo</surname><given-names>Takanobu</given-names></name><role content-type="http://credit.casrai.org/">Conceptualization</role><role content-type="http://credit.casrai.org/">Methodology</role><role content-type="http://credit.casrai.org/">Supervision</role><xref ref-type="aff" rid="aff008"><sup>8</sup></xref></contrib><contrib contrib-type="author"><name><surname>Noda</surname><given-names>Takeshi</given-names></name><role content-type="http://credit.casrai.org/">Data curation</role><role content-type="http://credit.casrai.org/">Supervision</role><role content-type="http://credit.casrai.org/">Validation</role><role content-type="http://credit.casrai.org/">Writing – original draft</role><role content-type="http://credit.casrai.org/">Writing – review & editing</role><xref ref-type="aff" rid="aff001"><sup>1</sup></xref><xref ref-type="aff" rid="aff002"><sup>2</sup></xref><xref ref-type="corresp" rid="cor001">*</xref></contrib></contrib-group><aff id="aff001"><label>1</label><addr-line>Center for Frontier Oral Science, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></aff><aff id="aff002"><label>2</label><addr-line>Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Suita, Osaka, Japan</addr-line></aff><aff id="aff003"><label>3</label><addr-line>China National Research Institute of Food and Fermentation Industries, Beijing, China</addr-line></aff><aff id="aff004"><label>4</label><addr-line>Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan</addr-line></aff><aff id="aff005"><label>5</label><addr-line>Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan</addr-line></aff><aff id="aff006"><label>6</label><addr-line>Medicinal Botanical Garden, Kobe Pharmaceutical University, Kobe, Hyogo, Japan</addr-line></aff><aff id="aff007"><label>7</label><addr-line>Laboratory of Medicinal Cell Biology, Kobe Pharmaceutical University, Kobe, Hyogo, Japan</addr-line></aff><aff id="aff008"><label>8</label><addr-line>Department of Molecular and Genetic Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Trajkovic</surname><given-names>Vladimir</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Faculty of Medicine, University of Belgrade, SERBIA</addr-line></aff><author-notes><fn fn-type="COI-statement" id="coi001"><p><bold>Competing Interests: </bold>Takeshi NODA has received research grant from Tsumura CO. LTD. Tsumura CO LTD has no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and decision to submit for publication. This research is not related to any of employment, consultancy, patents, products in development of Tsumura CO. LTD. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. The other authors have nothing to disclose.</p></fn><corresp id="cor001">* E-mail: <email>takenoda@dent.osaka-u.ac.jp</email></corresp></author-notes><pub-date pub-type="epub"><day>5</day><month>3</month><year>2020</year></pub-date><pub-date pub-type="collection"><year>2020</year></pub-date><volume>15</volume><issue>3</issue><elocation-id>e0230156</elocation-id><history><date date-type="received"><day>23</day><month>10</month><year>2019</year></date><date date-type="accepted"><day>22</day><month>2</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 Ikari et al</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Ikari et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="pone.0230156.pdf"></self-uri><abstract><p>Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca<sup>2+</sup> dependent manner. Cytosolic Ca<sup>2+</sup> concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca<sup>2+</sup> increase, thereby suppressing induction of autophagy.</p></abstract><funding-group><award-group id="award001"><funding-source><institution-wrap><institution-id institution-id-type="funder-id">http://dx.doi.org/10.13039/100008732</institution-id><institution>Uehara Memorial Foundation</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Noda</surname><given-names>Takeshi</given-names></name></principal-award-recipient></award-group><award-group id="award002"><funding-source><institution-wrap><institution-id institution-id-type="funder-id">http://dx.doi.org/10.13039/501100013420</institution-id><institution>Tsumura and Company</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Noda</surname><given-names>Takeshi</given-names></name></principal-award-recipient></award-group><funding-statement>Takeshi NODA has received research grant from Tsumura CO. LTD. Tsumura CO LTD has no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and decision to submit for publication.</funding-statement></funding-group><counts><fig-count count="8"></fig-count><table-count count="0"></table-count><page-count count="22"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>All relevant data are within the paper and its Supporting Information files.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>All relevant data are within the paper and its Supporting Information files.</p></notes></front><sub-article id="pone.0230156.r001" article-type="aggregated-review-documents"><front-stub><article-id pub-id-type="doi">10.1371/journal.pone.0230156.r001</article-id><title-group><article-title>Decision Letter 0</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Trajkovic</surname><given-names>Vladimir</given-names></name><role>Academic Editor</role></contrib></contrib-group><permissions><copyright-statement>© 2020 Vladimir Trajkovic</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Vladimir Trajkovic</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><related-article id="rel-obj001" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230156" related-article-type="reviewed-article"></related-article><custom-meta-group><custom-meta><meta-name>Submission Version</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></front-stub><body><p><named-content content-type="letter-date">2 Dec 2019</named-content></p><p>PONE-D-19-29517</p><p>Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.</p><p>PLOS ONE</p><p>Dear Dr. Noda,</p><p>Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.</p><p>Academic Editor’s comments:</p><p>1) Why there is no LC3-I band in the LC3 blot? Considering the proximity of LC3-I and LC3-II bands, how can the authors be sure that the visible band is LC3-II?</p><p>2) Total 4EBP1 should be analyzed together with phosphor-4EBP1.</p><p>Reviewer 1:</p><p>This manuscript by Ikari et al. describes the effects of TJ-35, a Kampo medicine herbal extract, on autophagy. Upon starvation, TFEB and ULK dephosphorylation, as well as calcium concentration influx, were suppressed in the presence of TJ-35. While these observations were interesting and potentially provide mechanistic insight on the action of TJ-35, there are some concerns that need to be addressed.</p><p>1. All image quantification should be described in more detail: e.g. how many experiments were performed, how many cells were counted and quantified…</p><p>2. ANOVA statistical analysis should be used for experimental groups >2 in figs 2, 3, 4, 5, 7, 8.</p><p>3. Many control experiments were missing. e.g. Fig 2A, 2B should include DMEM+TJ-35, Fig 2C should include no treatment, as well as Bafilomycin A treatment alone; Figs 3A, 3B, 3C, 4A need DMEM+TJ-35. These controls (or experimental) are important and can provide some useful information, especially considering TJ-35 was identified to have effects on tf-LC3 GFP/RFP ratio under medium rich (no starvation) condition in Fig 1B.</p><p>4. Fig 2A, could the authors provide the RFP image (of tf-LC3) on each panel and analyze the ratio of GFP/RFP as well? It can provide more information on which step autophagy is affected by TJ-35.</p><p>5. Fig 4C, it’s clear that TJ-35 had effect on TFEB dephosphorylation (based on the mobility shift). However, the effect on p-ULK1 was less obvious. It should be normalized with total ULK1 (the amount of ULK1 was not equal among the samples).</p><p>6. The title on Fig 8 legend is not appropriate. “TJ-35 prevents starvation induced calcium influx from the ER mediated by the IP3 receptor”, while this set of experiments did not involve TJ-35 at all. </p><p>7. In Fig 8, 0.5 micro M of EDTA was used to chelate calcium to conclude that the medium (or EBSS) was not the source of starvation-induced calcium influx. However, the concentration of calcium in EBSS and DMEM is at the range of milli M, the EDTA added was not sufficient to chelate all the calcium. Higher concentration of EDTA would need to be used for this experiment. </p><p>8. In Fig 6, have the authors tested if addition of TJ-35 has similar effect as cyclosporin A on inhibiting TFEB dephosphorylation through calcineurin? Similarly in Fig 7A, have the authors tested DMEM + ionomycin + TJ-35 condition?</p><p>Reviewer 2:</p><p>The report presents an interesting observation that “Shigyakusan” inhibits starvation-induced autophagy by promoting calcium dependent TFEB phosphorylation. Particularly, the elevation of cytosolic calcium upon starvation and involvement of ER calcium export to cytosol is exciting. The findings could be of interest to basic research in autophagy. However, more works needs to be carried out, this study could promise a discovery of novel autophagy modulator. For now, there are number of aspects that needs to be clarified before presentation in the PLOS One. Once these concerns are addressed, I recommend publication of this paper in PLOS One.</p><p>Major/Minor concerns are listed as follows.</p><p>1) Authors should include the exact ratio in which the different ingredients of “Kampo” (at least of the Shigyakusan/TJ-35) has been prepared.</p><p>2) Authors should explain on why they came up with certain concentration of positive control (Torin-1), negative control (Bafilomycin A) of autophagy for screening purpose. Importantly, author should explain how they choose specified concentration of each “Kampo” and how they were able to titrate its effect in relation to positive and negative control.</p><p>3) Representative images of at least for Control, Torin-1, BafilomycinA and TJ-35 should be included from the screening results.</p><p>4) After screening, authors already have an idea that autophagosome numbers might be decreased by TJ-35, therefore, authors should compare its effect with known inhibitors of autophagosome formation (possibly quantifying LC3 puncta). This is important to classify the effect of TJ-35 on autophagosome biogenesis. Comparing TJ-35 with the agents blocking autophagosome-lysosome fusion or agents preventing lysosomal acidification would only provide limited information that TJ-35 is an autophgay inhibitor as GFP/RFP intensity ratio is not affected by autophagosome population. Alternatively, authors should better show the GFP and RFP puncta per cell at least for Control, TJ-35, Torin-1, and Bafilomycin A.</p><p>5) Combined effect of TJ-35 with Chloroquine or Bafilomycin A is needed to suggest that TJ-35 prevent autophagosome formation. (Possibly TJ-35 pre-treatment followed by addition of chloroquine or Bafilomycin A).</p><p>6) Is this effect of TJ-35 limited to starvation-induced autophagy?</p><p>7) Why there is a difference in the band size of TFEB between 4th lane (DMEM+TJ-35) and 5th lane (EBSS+TJ-35) in the Figure 4C? In another word, is phosphorylation state of TFEB is different in DMEM+TJ-35 and EBSS+TJ-35? Overall, the blot shown in Figure 4C is confusing as authors claim that the difference in the size of TFEB represent its phosphorylation state? Is it because of shorter exposure time that both Phospho-TFEB and TFEB is not visible in this blot as shown in Figure 6 (Clearly, TFEB antibody could detect both phospho TFEB and TFEB)? Authors should either show the clear TFEB bands in the Figure 4C or perform cell fraction to show endogenous TFEB in cytosol or nucleus? Authors should explain, why there is strong dephosphorylation state of TFEB (if it is), in the 6th lane as compared with 3rd and 5th lane.</p><p>8) Why 4EBP1 band size shown in the Figure 4C is different between samples (especially lane 4th, 5th and 6th? Does this antibody detect both phospho-EBP1 and total EBP1?</p><p>9) Why there is discrepancy in the Ulk1 and S6k phosphorylation by TJ-35 in DMEM and EBSS as shown in Figure 4C? Could this data suggest that activity of TJ-35 on mTOR is dependent upon nutrient availability?</p><p>10) Authors should explain the content of the reference# 35 mentioned in the line#349, page 14.</p><p>11) How Inonomycin and cyclosporin A affect autophagy flux and mTOR activity? It has been reported that Ionomycin results in massive accumulation of autophagosome (Hansen M et al., Mol Cell 2007), could be a characteristic of impaired autophagy. It is important to show that how ionomycin and cyclosporinA affects autophagy flux by using tf-LC3 system. Could they do an assay to compare the effect of Ionomycin and Cyclosporin A with TJ-35, this could be important for the claim that TJ-35 inhibits calcium dependent autophagosome formation. </p><p>12) To support the conclusion drawn from Figure 8A, authors should include a calcium free medium in their experiment.</p><p>13) I believe that it should be “calcium efflux from the ER” not “calcium influx from the ER” as mentioned in the line #412, page 16. Author should correct this wherever possible in the manuscript text and figure legend.</p><p>14) Authors should design an assay to conclude that the effect of the TJ-35 on autophagy activity requires all the five ingredients and try to explain why exact combination of all ingredient is essential. Alternatively, authors should perform assay to find out which ingredient of TJ-35 is most essential in modulating autophagy activity by applying the principle of exclusion and discuss the direction for identification/purification of novel molecules present in the ingredients of TJ-35 that could modulate calcium dependent autophagy.</p><p>==============================</p><p>We would appreciate receiving your revised manuscript by Jan 16 2020 11:59PM. When you are ready to submit your revision, log on to <ext-link ext-link-type="uri" xlink:href="https://www.editorialmanager.com/pone/">https://www.editorialmanager.com/pone/</ext-link> and select the 'Submissions Needing Revision' folder to locate your manuscript file.</p><p>If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.</p><p>To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: <ext-link ext-link-type="uri" xlink:href="http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols">http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols</ext-link></p><p>Please include the following items when submitting your revised manuscript:</p><p><list list-type="bullet"><list-item><p>A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.</p></list-item><list-item><p>A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.</p></list-item><list-item><p>An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.</p></list-item></list></p><p>Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.</p><p>We look forward to receiving your revised manuscript.</p><p>Kind regards,</p><p>Vladimir Trajkovic</p><p>Academic Editor</p><p>PLOS ONE</p><p>Journal Requirements:</p><p>When submitting your revision, we need you to address these additional requirements.</p><p>1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at</p><p><ext-link ext-link-type="uri" xlink:href="http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf">http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf">http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf</ext-link></p><p>2. We understand that you obtained the Kampo medicines from Tsumura (Tokyo, Japan) for this study. For purposes of reporting, we request that you provide additional details as to the source of this material (please see <ext-link ext-link-type="uri" xlink:href="http://journals.plos.org/plosone/s/criteria-for-publication#loc-3">http://journals.plos.org/plosone/s/criteria-for-publication#loc-3</ext-link> for more information). Please provide the following details: the specific medicines obtained from Tsumura, including the product numbers and any lot numbers provided in your Methods section. In addition, please clarify whether the medicines obtained from Tsumura came with any chemical assessments and quality assessments.</p><p>3. Please provide additional information about each of the cell lines used in this work, including source and any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see <ext-link ext-link-type="uri" xlink:href="http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines.">" ext-link-type="uri" xlink:type="simple">http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines."</ext-link></p><p>4. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see <ext-link ext-link-type="uri" xlink:href="https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting">https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting</ext-link>.</p><p>5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at <ext-link ext-link-type="uri" xlink:href="https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements">https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements</ext-link> and <ext-link ext-link-type="uri" xlink:href="https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files">https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files</ext-link>. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: <ext-link ext-link-type="uri" xlink:href="https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels">https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels</ext-link>.</p><p>In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at <email>plosone@plos.org</email> if you have any questions.</p><p>6. Thank you for stating the following in the Financial Disclosure section:</p><p>'TN.</p><p>This work was supported by a grant from Uehara Memorial Foundation (TN) and commissioned work from the Tsumura Corporation (Tokyo, Japan). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.'</p><p>We note that you received funding from a commercial source: Tsumura Corporation</p><p>a. Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other <ext-link ext-link-type="uri" xlink:href="http://www.plosone.org/static/competing.action">relevant declarations</ext-link> relating to employment, consultancy, patents, products in development, marketed products, etc.</p><p>Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors <ext-link ext-link-type="uri" xlink:href="http://journals.plos.org/plosone/s/competing-interests">http://journals.plos.org/plosone/s/competing-interests</ext-link>). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.</p><p>b. Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf.</p><p>Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: <ext-link ext-link-type="uri" xlink:href="http://journals.plos.org/plosone/s/competing-interests">http://journals.plos.org/plosone/s/competing-interests</ext-link></p><p>[Note: HTML markup is below. Please do not edit.]</p><p>Reviewers' comments:</p><p>Reviewer's Responses to Questions</p><p><bold>Comments to the Author</bold></p><p>1. Is the manuscript technically sound, and do the data support the conclusions?</p><p>The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. </p><p>Reviewer #1: Partly</p><p>Reviewer #2: Yes</p><p>**********</p><p>2. Has the statistical analysis been performed appropriately and rigorously? </p><p>Reviewer #1: No</p><p>Reviewer #2: Yes</p><p>**********</p><p>3. Have the authors made all data underlying the findings in their manuscript fully available?</p><p>The <ext-link ext-link-type="uri" xlink:href="http://www.plosone.org/static/policies.action#sharing">PLOS Data policy</ext-link> requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.</p><p>Reviewer #1: Yes</p><p>Reviewer #2: No</p><p>**********</p><p>4. Is the manuscript presented in an intelligible fashion and written in standard English?</p><p>PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.</p><p>Reviewer #1: Yes</p><p>Reviewer #2: Yes</p><p>**********</p><p>5. Review Comments to the Author</p><p>Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)</p><p>Reviewer #1: This manuscript by Ikari et al. describes the effects of TJ-35, a Kampo medicine herbal extract, on autophagy. Upon starvation, TFEB and ULK dephosphorylation, as well as calcium concentration influx, were suppressed in the presence of TJ-35. While these observations were interesting and potentially provide mechanistic insight on the action of TJ-35, there are some concerns that need to be addressed.</p><p>1. All image quantification should be described in more detail: e.g. how many experiments were performed, how many cells were counted and quantified,…</p><p>2. ANOVA statistical analysis should be used for experimental groups 2 in figs 2, 3, 4, 5, 7, 8.</p><p>3. Many control experiments were missing. e.g. Fig 2A, 2B should include DMEM+TJ-35, Fig 2C should include no treatment, as well as Bafilomycin A treatment alone; Figs 3A, 3B, 3C, 4A need DMEM+TJ-35. These controls (or experimental) are important and can provide some useful information, especially considering TJ-35 was identified to have effects on tf-LC3 GFP/RFP ratio under medium rich (no starvation) condition in Fig 1B.</p><p>4. Fig 2A, could the authors provide the RFP image (of tf-LC3) on each panel and analyze the ratio of GFP/RFP as well? It can provide more information on which step autophagy is affected by TJ-35.</p><p>5. Fig 4C, it’s clear that TJ-35 had effect on TFEB dephosphorylation (based on the mobility shift). However, the effect on p-ULK1 was less obvious. It should be normalized with total ULK1 (the amount of ULK1 was not equal among the samples).</p><p>6. The title on Fig 8 legend is not appropriate. “TJ-35 prevents starvation induced calcium influx from the ER mediated by the IP3 receptor”, while this set of experiments did not involve TJ-35 at all.</p><p>7. In Fig 8, 0.5 micro M of EDTA was used to chelate calcium to conclude that the medium (or EBSS) was not the source of starvation-induced calcium influx. However, the concentration of calcium in EBSS and DMEM is at the range of milli M, the EDTA added was not sufficient to chelate all the calcium. Higher concentration of EDTA would need to be used for this experiment.</p><p>8. In Fig 6, have the authors tested if addition of TJ-35 has similar effect as cyclosporin A on inhibiting TFEB dephosphorylation through calcineurin? Similarly in Fig 7A, have the authors tested DMEM + ionomycin + TJ-35 condition?</p><p>Reviewer #2: The report presents an interesting observation that “Shigyakusan” inhibits starvation-induced autophagy by promoting calcium dependent TFEB phosphorylation. Particularly, the elevation of cytosolic calcium upon starvation and involvement of ER calcium export to cytosol is exciting. The findings could be of interest to basic research in autophagy. However, more works needs to be carried out, this study could promise a discovery of novel autophagy modulator. For now, there are number of aspects that needs to be clarified before presentation in the PLOS One. Once these concerns are addressed, I recommend publication of this paper in PLOS One.</p><p>Major/Minor concerns are listed as follows.</p><p>1) Authors should include the exact ratio in which the different ingredients of “Kampo” (at least of the Shigyakusan/TJ-</p><p>35) has been prepared.</p><p>2) Authors should explain on why they came up with certain concentration of positive control (Torin-1), negative control</p><p>(Bafilomycin A) of autophagy for screening purpose. Importantly, author should explain how they choose specified</p><p>concentration of each “Kampo” and how they were able to titrate its effect in relation to positive and negative control.</p><p>3) Representative images of at least for Control, Torin-1, BafilomycinA and TJ-35 should be included from the screening</p><p>results.</p><p>4) After screening, authors already have an idea that autophagosome numbers might be decreased by TJ-35, therefore,</p><p>authors should compare its effect with known inhibitors of autophagosome formation (possibly quantifying LC3 puncta).</p><p>This is important to classify the effect of TJ-35 on autophagosome biogenesis. Comparing TJ-35 with the agents blocking</p><p>autophagosome-lysosome fusion or agents preventing lysosomal acidification would only provide limited information</p><p>that TJ-35 is an autophgay inhibitor as GFP/RFP intensity ratio is not affected by autophagosome population.</p><p>Alternatively, authors should better show the GFP and RFP puncta per cell at least for Control, TJ-35, Torin-1, and</p><p>Bafilomycin A.</p><p>5) Combined effect of TJ-35 with Chloroquine or Bafilomycin A is needed to suggest that TJ-35 prevent autophagosome</p><p>formation. (Possibly TJ-35 pre-treatment followed by addition of chloroquine or Bafilomycin A).</p><p>6) Is this effect of TJ-35 limited to starvation-induced autophagy?</p><p>7) Why there is a difference in the band size of TFEB between 4th lane (DMEM+TJ-35) and 5th lane (EBSS+TJ-35) in the</p><p>Figure 4C? In another word, is phosphorylation state of TFEB is different in DMEM+TJ-35 and EBSS+TJ-35? Overall, the</p><p>blot shown in Figure 4C is confusing as authors claim that the difference in the size of TFEB represent its</p><p>phosphorylation state? Is it because of shorter exposure time that both Phospho-TFEB and TFEB is not visible in this blot</p><p>as shown in Figure 6 (Clearly, TFEB antibody could detect both phospho TFEB and TFEB)? Authors should either show</p><p>the clear TFEB bands in the Figure 4C or perform cell fraction to show endogenous TFEB in cytosol or nucleus? Authors</p><p>should explain, why there is strong dephosphorylation state of TFEB (if it is), in the 6th lane as compared with 3rd and</p><p>5th lane.</p><p>8) Why 4EBP1 band size shown in the Figure 4C is different between samples (especially lane 4th, 5th and 6th? Does this</p><p>antibody detect both phospho-EBP1 and total EBP1?</p><p>9) Why there is discrepancy in the Ulk1 and S6k phosphorylation by TJ-35 in DMEM and EBSS as shown in Figure 4C?</p><p>Could this data suggest that activity of TJ-35 on mTOR is dependent upon nutrient availability?</p><p>10) Authors should explain the content of the reference# 35 mentioned in the line#349, page 14.</p><p>11) How Inonomycin and cyclosporin A affect autophagy flux and mTOR activity? It has been reported that Ionomycin</p><p>results in massive accumulation of autophagosome (Hansen M et al., Mol Cell 2007), could be a characteristic of</p><p>impaired autophagy. It is important to show that how ionomycin and cyclosporinA affects autophagy flux by using tf-</p><p>LC3 system. Could they do an assay to compare the effect of Ionomycin and Cyclosporin A with TJ-35, this could be</p><p>important for the claim that TJ-35 inhibits calcium dependent autophagosome formation.</p><p>12) To support the conclusion drawn from Figure 8A, authors should include a calcium free medium</p><p>in their experiment.</p><p>13) I believe that it should be “calcium efflux from the ER” not “calcium influx from the ER” as mentioned in the line #412,</p><p>page 16. Author should correct this wherever possible in the manuscript text and figure legend.</p><p>14) Authors should design an assay to conclude that the effect of the TJ-35 on autophagy activity requires all the five</p><p>ingredients and try to explain why exact combination of all ingredient is essential. Alternatively, authors should</p><p>perform assay to find out which ingredient of TJ-35 is most essential in modulating autophagy activity by applying the</p><p>principle of exclusion and discuss the direction for identification/purification of novel molecules present in the</p><p>ingredients of TJ-35 that could modulate calcium dependent autophagy.</p><p>**********</p><p>6. PLOS authors have the option to publish the peer review history of their article (<ext-link ext-link-type="uri" xlink:href="https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history">what does this mean?</ext-link>). If published, this will include your full peer review and any attached files.</p><p>If you choose “no”, your identity will remain anonymous but your review may still be made public.</p><p><bold>Do you want your identity to be public for this peer review?</bold> For information about this choice, including consent withdrawal, please see our <ext-link ext-link-type="uri" xlink:href="https://www.plos.org/privacy-policy">Privacy Policy</ext-link>.</p><p>Reviewer #1: No</p><p>Reviewer #2: No</p><p>[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]</p><p>While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, <ext-link ext-link-type="uri" xlink:href="https://pacev2.apexcovantage.com/">https://pacev2.apexcovantage.com/</ext-link>. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at <email>figures@plos.org</email>. Please note that Supporting Information files do not need this step.</p></body></sub-article><sub-article id="pone.0230156.r002" article-type="author-comment"><front-stub><article-id pub-id-type="doi">10.1371/journal.pone.0230156.r002</article-id><title-group><article-title>Author response to Decision Letter 0</article-title></title-group><related-article id="rel-obj002" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230156" related-article-type="editor-report"></related-article><custom-meta-group><custom-meta><meta-name>Submission Version</meta-name><meta-value>1</meta-value></custom-meta></custom-meta-group></front-stub><body><p><named-content content-type="author-response-date">20 Jan 2020</named-content></p><p>I have included this information in cover letter with attaching several figures.</p><p>January 17, 2020</p><p>MS ID#: PONE-D-19-29517</p><p>Dr. Joerg Heber,</p><p>Editor-in-Chief </p><p>PLoS One</p><p>Dear Dr. Heber:</p><p>Please find enclosed the revised version of our manuscript, “Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan”. We have responded to all concerns raised by the reviewers by performing a number of experiments. </p><p>Major changes from the previous version are: i) evaluation of the effects of each herbal ingredient (supplemental figure 4), ii) evaluation of the aggrephagy (Supplemental Figure 2), iii) effect of cyclosporin and ionomycin on autophagy (Supplemental Figure 7). We thank the reviewers for their constructive comments, due to which the quality of our data has improved.</p><p>We hope that these revisions are sufficient to make our manuscript suitable for publication in PLoS One.</p><p>Competing interests: Takeshi NODA has received research grant from Tsumura CO. LTD. Tsumura CO LTD has no role in the study design; collection, analysis, and interpretation of data; writing of the paper; and decision to submit for publication. This research is not related to any of employment, consultancy, patents, products in development of Tsumura CO. LTD. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. The other authors have nothing to disclose.</p><p>We have attached our point-by-point answers to the comments of the reviewers below.</p><p>Thank you for your consideration. </p><p>With regards,</p><p>Takeshi NODA, Ph.D. Professor,</p><p>Center for Frontier Oral Science, </p><p>Graduate School of Dentistry, Osaka University</p><p>1-8 Yamadaoka, Suita, Osaka 565-0871, Japan</p><p>E-mail: <email>takenoda@dent.osaka-u.ac.jp</email></p><p>Tel: +81-6-6879-2976</p><p>Fax: +81-6-6879-2110</p><p>Academic Editor’s comments:</p><p>1) Why there is no LC3-I band in the LC3 blot? Considering the proximity of LC3-I and LC3-II bands, how can the authors be sure that the visible band is LC3-II?</p><p>Answer</p><p>HeLa cells are known to express less LC3-I. To substantiate this point, we compared the band pattern with the sample from MEF cells, which express more LC3-I. As shown in supplemental figure 3, the bands correspond to the LC3-II form but not LC3-I form.</p><p>2) Total 4EBP1 should be analyzed together with phosphor-4EBP1.</p><p>Answer</p><p>We had shown total 4EBP1 with anti-4EBP1 antibody. Dephosphorylation is indicated by the down-shift of the band size.</p><p>Reviewer 1:</p><p>This manuscript by Ikari et al. describes the effects of TJ-35, a Kampo medicine herbal extract, on autophagy. Upon starvation, TFEB and ULK dephosphorylation, as well as calcium concentration influx, were suppressed in the presence of TJ-35. While these observations were interesting and potentially provide mechanistic insight on the action of TJ-35, there are some concerns that need to be addressed.</p><p>1. All image quantification should be described in more detail: e.g. how many experiments were performed, how many cells were counted and quantified…</p><p>Answer</p><p>We have added the required information to the corresponding figure legends.</p><p>2. ANOVA statistical analysis should be used for experimental groups >2 in figs 2, 3, 4, 5, 7, 8.</p><p>Answer</p><p>We aimed to show the suppressive effect of TJ-35 only in the starvation condition; thus, we understand that it will be appropriate to use the t-test for comparison between 2 groups.</p><p>3. Many control experiments were missing. e.g. Fig 2A, 2B should include DMEM+TJ-35, Fig 2C should include no treatment, as well as Bafilomycin A treatment alone; Figs 3A, 3B, 3C, 4A need DMEM+TJ-35. These controls (or experimental) are important and can provide some useful information, especially considering TJ-35 was identified to have effects on tf-LC3 GFP/RFP ratio under medium rich (no starvation) condition in Fig 1B.</p><p>Answer</p><p>According to this comment, we added control data in Figs. 2, 3. and 4. Especially, in Fig. 2C, we could not observe a significant effect in DMEM. It is possible that the 6-hour treatment may not be enough for the effect in DMEM. We will investigate this in our future studies.</p><p>4. Fig 2A, could the authors provide the RFP image (of tf-LC3) on each panel and analyze the ratio of GFP/RFP as well? It can provide more information on which step autophagy is affected by TJ-35.</p><p>Answer</p><p>We have added RFP image and the signal ratio in Fig 2A. These data indicate that TJ-35 suppresses autophagosome and autolysosome induction. We examined which step is affected in the following experiments.</p><p>5. Fig 4C, it’s clear that TJ-35 had effect on TFEB dephosphorylation (based on the mobility shift). However, the effect on p-ULK1 was less obvious. It should be normalized with total ULK1 (the amount of ULK1 was not equal among the samples).</p><p>Answer</p><p>We here provided quantification data of ULK1 and phosphor-ULK1 and tubline. And also ULK1 blot was replaced with the same data but whose contrast is less exagerated. As suggested by the reviewer, the effect on ULK1 might be less noticeale. We still cannot provide the mechanism through which ULK1 phosphorylation is affected by TJ-35 and need to examine this in our future studies.</p><p>6. The title on Fig 8 legend is not appropriate. “TJ-35 prevents starvation induced calcium influx from the ER mediated by the IP3 receptor”, while this set of experiments did not involve TJ-35 at all. </p><p>Answer</p><p>According to this comment, we amended the title as follows: “Starvation induced calcium efflux from the ER mediated by the IP3 receptor”.</p><p>7. In Fig 8, 0.5 micro M of EDTA was used to chelate calcium to conclude that the medium (or EBSS) was not the source of starvation-induced calcium influx. However, the concentration of calcium in EBSS and DMEM is at the range of milli M, the EDTA added was not sufficient to chelate all the calcium. Higher concentration of EDTA would need to be used for this experiment. </p><p>Answer</p><p>Thanks for this comment. We noticed our mistake and have now amended it.</p><p>8. In Fig 6, have the authors tested if addition of TJ-35 has similar effect as cyclosporin A on inhibiting TFEB dephosphorylation through calcineurin? Similarly in Fig 7A, have the authors tested DMEM + ionomycin + TJ-35 condition?</p><p>Answer</p><p>As shown in Fig. 6, TJ-35 did not exert a significant suppressive effect on the dephosphorylation of TFEB and nuclear translocation following ionomycin administration. However, due to an unknown reason, the combination of ionomycin and TJ-35 seems to affect the TFEB protein level. Since we cannot pursue this point in the current study, we will do so in a future study.</p><p>Reviewer 2:</p><p>The report presents an interesting observation that “Shigyakusan” inhibits starvation-induced autophagy by promoting calcium dependent TFEB phosphorylation. Particularly, the elevation of cytosolic calcium upon starvation and involvement of ER calcium export to cytosol is exciting. The findings could be of interest to basic research in autophagy. However, more works needs to be carried out, this study could promise a discovery of novel autophagy modulator. For now, there are number of aspects that needs to be clarified before presentation in the PLOS One. Once these concerns are addressed, I recommend publication of this paper in PLOS One.</p><p>Major/Minor concerns are listed as follows.</p><p>1) Authors should include the exact ratio in which the different ingredients of “Kampo” (at least of the Shigyakusan/TJ-35) has been prepared.</p><p>Answer</p><p>According to this comment, we described the ingredient ratio of Shigyakusan in the Materials and Methods section.</p><p>2) Authors should explain on why they came up with certain concentration of positive control (Torin-1), negative control (Bafilomycin A) of autophagy for screening purpose. Importantly, author should explain how they choose specified concentration of each “Kampo” and how they were able to titrate its effect in relation to positive and negative control.</p><p>Answer</p><p>We have added the following sentences on page 10, line 244</p><p>“To determine Torin-1 and Bafilomycin A concentration, we followed standard treatment conditions described previously [21, 22].</p><p>For determining Kampo concentration, we pre-screened small scale samples (20 samples) with different concentrations (100, 200, 400 �g/ml) and the concentration of 400 �g/ml exerted the most prominent effects; accordingly, we adopted it.”</p><p>3) Representative images of at least for Control, Torin-1, BafilomycinA and TJ-35 should be included from the screening results.</p><p>Answer</p><p>We have added the representative images in figure 1C.</p><p>4) After screening, authors already have an idea that autophagosome numbers might be decreased by TJ-35, therefore, authors should compare its effect with known inhibitors of autophagosome formation (possibly quantifying LC3 puncta). This is important to classify the effect of TJ-35 on autophagosome biogenesis. Comparing TJ-35 with the agents blocking autophagosome-lysosome fusion or agents preventing lysosomal acidification would only provide limited information that TJ-35 is an autophgay inhibitor as GFP/RFP intensity ratio is not affected by autophagosome population. Alternatively, authors should better show the GFP and RFP puncta per cell at least for Control, TJ-35, Torin-1, and Bafilomycin A.</p><p>Answer</p><p>After screening, we postulated two possibilities, i.e., formation of autophagosome is suppressed or fusion/degradation is suppressed. If the former is correct, the latter need not be tested further. We examined several standard markers of autophagosome formation including Atg5, WIPI1, and ULK1, and all the resulting data indicated that autophagosome formation was suppressed. In the revised text, as suggested by the reviewer in comment 5, we added new data that LC3 puncta formation is suppressed by TJ-35 treatment even if autophagosome fusion/degradation is suppressed by bafilomycin treatment (supplemental figure 5). This also clearly supports that autophagosome formation is suppressed. </p><p>5) Combined effect of TJ-35 with Chloroquine or Bafilomycin A is needed to suggest that TJ-35 prevent autophagosome formation. (Possibly TJ-35 pre-treatment followed by addition of chloroquine or Bafilomycin A).</p><p>Answer</p><p>As mentioned above, we added this experiment in supplemental figure 5, and it supports that autophagosome formation is suppressed by TJ-35 treatment.</p><p>6) Is this effect of TJ-35 limited to starvation-induced autophagy?</p><p>Answer</p><p>Among the several types of autophagy, we further examined if TJ-35 affects aggrephagy, which targets protein aggregation. TJ-35 did not affect the efficiency of aggrephagy (supplemental figure 2). Therefore, we speculate that TJ-35 did not affect the broad range of autophagy.</p><p>7) Why there is a difference in the band size of TFEB between 4th lane (DMEM+TJ-35) and 5th lane (EBSS+TJ-35) in the Figure 4C? In another word, is phosphorylation state of TFEB is different in DMEM+TJ-35 and EBSS+TJ-35? Overall, the blot shown in Figure 4C is confusing as authors claim that the difference in the size of TFEB represent its phosphorylation state? Is it because of shorter exposure time that both Phospho-TFEB and TFEB is not visible in this blot as shown in Figure 6 (Clearly, TFEB antibody could detect both phospho TFEB and TFEB)? Authors should either show the clear TFEB bands in the Figure 4C or perform cell fraction to show endogenous TFEB in cytosol or nucleus? Authors should explain, why there is strong dephosphorylation state of TFEB (if it is), in the 6th lane as compared Anwith 3rd and 5th lane.</p><p>Answer</p><p>Due to band intensity fluctuation, there may have been some confusion. We repeated the experiments, and succeeded in obtaining clearer and even intensity band profiles in figure 4D.</p><p>8) Why 4EBP1 band size shown in the Figure 4C is different between samples (especially lane 4th, 5th and 6th? Does this antibody detect both phospho-EBP1 and total EBP1?</p><p>Answer</p><p>We are sorry for the shortcoming of the explanation. We added the following sentences on page 14, Line 360.</p><p>“Western blotting with anti-translation initiation factor 4E-binding protein (4EBP1) antibody reacted with both phosphorylated and unphosphorylated 4EBP1, with the latter represented by the down-shift of the band size. Dephosphorylation of 4EBP1 was also unaffected by TJ-35 (Fig 4E).”</p><p>9) Why there is discrepancy in the Ulk1 and S6k phosphorylation by TJ-35 in DMEM and EBSS as shown in Figure 4C? Could this data suggest that activity of TJ-35 on mTOR is dependent upon nutrient availability?</p><p>Answer</p><p>Regarding S6k and 4EBP1, TJ-35 demonstrated no effect on its phosphorylation status by mTOR inactivation during starvation. We therefore suggest that TJ-35 effect is independent of mTOR activity.</p><p>10) Authors should explain the content of the reference# 35 mentioned in the line#349, page 14.</p><p>Answer</p><p>Line #349 would be referring to reference #34, not #35 of the original manuscript. We referred to this paper (current #37) citing the physical interaction between s6k and mTOR.</p><p>11) How Inonomycin and cyclosporin A affect autophagy flux and mTOR activity? It has been reported that Ionomycin results in massive accumulation of autophagosome (Hansen M et al., Mol Cell 2007), could be a characteristic of impaired autophagy. It is important to show that how ionomycin and cyclosporinA affects autophagy flux by using tf-LC3 system. Could they do an assay to compare the effect of Ionomycin and Cyclosporin A with TJ-35, this could be important for the claim that TJ-35 inhibits calcium dependent autophagosome formation.</p><p>According to this comment, we treated the cells with ionomycin and cyclosporin A to observe if any effects could be observed on autophagy. Hansen M et al, treated the cells with ionomycin for 24 hours, but it was highly toxic to cells, and hence, we treated them for 30 min. Ionomycin treatment in DMEM resulted in accumulation of GFP-LC3 similar to Hansen M, supporting a Ca-dependent autophagy induction mechanism. In addition, cyclosporin A treatment in EBSS reduced GFP-LC3 formation, supporting the positive role of calcineurin in autophagy induction. We added these data as supplemental figure 7. </p><p>12) To support the conclusion drawn from Figure 8A, authors should include a calcium free medium in their experiment.</p><p>Answer</p><p>According to this comment, we used calcium free HBSS medium. As shown here, [Ca] still increased, supporting our conclusion that calcium is released from the intracellular pool. However, that condition seems to be relatively toxic to cells judged from cell morphology; therefore, we would not show this in the manuscript.</p><p>13) I believe that it should be “calcium efflux from the ER” not “calcium influx from the ER” as mentioned in the line #412, page 16. Author should correct this wherever possible in the manuscript text and figure legend.</p><p>Answer</p><p>According to this comment, we have corrected as “calcium efflux from the ER” in the line #475, page 19</p><p>14) Authors should design an assay to conclude that the effect of the TJ-35 on autophagy activity requires all the five ingredients and try to explain why exact combination of all ingredient is essential. Alternatively, authors should perform assay to find out which ingredient of TJ-35 is most essential in modulating autophagy activity by applying the principle of exclusion and discuss the direction for identification/purification of novel molecules present in the ingredients of TJ-35 that could modulate calcium dependent autophagy.</p><p>According to this comment, we prepared TJ-35 mixtures lacking each of the four herbal ingredients and observed its effect on autophagy. Omitting any of the four ingredients resulted in failure of suppression of autophagy, supporting that their combination is critical. We added these data as supplemental figure 4 and described it on page 11, line 284.</p></body></sub-article><sub-article id="pone.0230156.r003" article-type="aggregated-review-documents"><front-stub><article-id pub-id-type="doi">10.1371/journal.pone.0230156.r003</article-id><title-group><article-title>Decision Letter 1</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Trajkovic</surname><given-names>Vladimir</given-names></name><role>Academic Editor</role></contrib></contrib-group><permissions><copyright-statement>© 2020 Vladimir Trajkovic</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Vladimir Trajkovic</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><related-article id="rel-obj003" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230156" related-article-type="reviewed-article"></related-article><custom-meta-group><custom-meta><meta-name>Submission Version</meta-name><meta-value>1</meta-value></custom-meta></custom-meta-group></front-stub><body><p><named-content content-type="letter-date">24 Feb 2020</named-content></p><p>Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.</p><p>PONE-D-19-29517R1</p><p>Dear Dr. Noda,</p><p>We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.</p><p>Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.</p><p>Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at <ext-link ext-link-type="uri" xlink:href="https://www.editorialmanager.com/pone/">https://www.editorialmanager.com/pone/</ext-link>, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at <email>authorbilling@plos.org</email>.</p><p>If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact <email>onepress@plos.org</email>.</p><p>With kind regards,</p><p>Vladimir Trajkovic</p><p>Academic Editor</p><p>PLOS ONE</p><p>Additional Editor Comments (optional):</p><p>Reviewers' comments:</p><p>Reviewer's Responses to Questions</p><p><bold>Comments to the Author</bold></p><p>1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.</p><p>Reviewer #1: All comments have been addressed</p><p>**********</p><p>2. Is the manuscript technically sound, and do the data support the conclusions?</p><p>The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. </p><p>Reviewer #1: Yes</p><p>**********</p><p>3. Has the statistical analysis been performed appropriately and rigorously? </p><p>Reviewer #1: Yes</p><p>**********</p><p>4. Have the authors made all data underlying the findings in their manuscript fully available?</p><p>The <ext-link ext-link-type="uri" xlink:href="http://www.plosone.org/static/policies.action#sharing">PLOS Data policy</ext-link> requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.</p><p>Reviewer #1: Yes</p><p>**********</p><p>5. Is the manuscript presented in an intelligible fashion and written in standard English?</p><p>PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.</p><p>Reviewer #1: Yes</p><p>**********</p><p>6. Review Comments to the Author</p><p>Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)</p><p>Reviewer #1: (No Response)</p><p>**********</p><p>7. PLOS authors have the option to publish the peer review history of their article (<ext-link ext-link-type="uri" xlink:href="https://journals.plos.org/plosone/s/editorial-and-peer-review-process#loc-peer-review-history">what does this mean?</ext-link>). If published, this will include your full peer review and any attached files.</p><p>If you choose “no”, your identity will remain anonymous but your review may still be made public.</p><p><bold>Do you want your identity to be public for this peer review?</bold> For information about this choice, including consent withdrawal, please see our <ext-link ext-link-type="uri" xlink:href="https://www.plos.org/privacy-policy">Privacy Policy</ext-link>.</p><p>Reviewer #1: No</p></body></sub-article><sub-article id="pone.0230156.r004" article-type="editor-report"><front-stub><article-id pub-id-type="doi">10.1371/journal.pone.0230156.r004</article-id><title-group><article-title>Acceptance letter</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Trajkovic</surname><given-names>Vladimir</given-names></name><role>Academic Editor</role></contrib></contrib-group><permissions><copyright-statement>© 2020 Vladimir Trajkovic</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Vladimir Trajkovic</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><related-article id="rel-obj004" ext-link-type="doi" xlink:href="10.1371/journal.pone.0230156" related-article-type="reviewed-article"></related-article></front-stub><body><p><named-content content-type="letter-date">26 Feb 2020</named-content></p><p>PONE-D-19-29517R1 </p><p>Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan </p><p>Dear Dr. Noda:</p><p>I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. </p><p>If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact <email>onepress@plos.org</email>.</p><p>For any other questions or concerns, please email <email>plosone@plos.org</email>. </p><p>Thank you for submitting your work to PLOS ONE.</p><p>With kind regards,</p><p>PLOS ONE Editorial Office Staff</p><p>on behalf of</p><p>Prof. Vladimir Trajkovic </p><p>Academic Editor</p><p>PLOS ONE</p></body></sub-article></pmc><affiliations><list><country><li>Japon</li><li>République populaire de Chine</li></country><settlement><li>Pékin</li></settlement></list><tree><country name="Japon"><noRegion><name sortKey="Ikari, Sumiko" sort="Ikari, Sumiko" uniqKey="Ikari S" first="Sumiko" last="Ikari">Sumiko Ikari</name></noRegion><name sortKey="Araki, Yasuhiro" sort="Araki, Yasuhiro" uniqKey="Araki Y" first="Yasuhiro" last="Araki">Yasuhiro Araki</name><name sortKey="Hao, Feike" sort="Hao, Feike" uniqKey="Hao F" first="Feike" last="Hao">Feike Hao</name><name sortKey="Imai, Kenta" sort="Imai, Kenta" uniqKey="Imai K" first="Kenta" last="Imai">Kenta Imai</name><name sortKey="Lu, Shiou Ling" sort="Lu, Shiou Ling" uniqKey="Lu S" first="Shiou-Ling" last="Lu">Shiou-Ling Lu</name><name sortKey="Nishiyama, Yumi" sort="Nishiyama, Yumi" uniqKey="Nishiyama Y" first="Yumi" last="Nishiyama">Yumi Nishiyama</name><name sortKey="Noda, Takeshi" sort="Noda, Takeshi" uniqKey="Noda T" first="Takeshi" last="Noda">Takeshi Noda</name><name sortKey="Noda, Takeshi" sort="Noda, Takeshi" uniqKey="Noda T" first="Takeshi" last="Noda">Takeshi Noda</name><name sortKey="Otomo, Takanobu" sort="Otomo, Takanobu" uniqKey="Otomo T" first="Takanobu" last="Otomo">Takanobu Otomo</name><name sortKey="Shitan, Nobukazu" sort="Shitan, Nobukazu" uniqKey="Shitan N" first="Nobukazu" last="Shitan">Nobukazu Shitan</name><name sortKey="Tsai, Chao Yuan" sort="Tsai, Chao Yuan" uniqKey="Tsai C" first="Chao-Yuan" last="Tsai">Chao-Yuan Tsai</name><name sortKey="Yamamoto, Yo Hei" sort="Yamamoto, Yo Hei" uniqKey="Yamamoto Y" first="Yo-Hei" last="Yamamoto">Yo-Hei Yamamoto</name><name sortKey="Yoshimori, Tamotsu" sort="Yoshimori, Tamotsu" uniqKey="Yoshimori T" first="Tamotsu" last="Yoshimori">Tamotsu Yoshimori</name></country><country name="République populaire de Chine"><noRegion><name sortKey="Hao, Feike" sort="Hao, Feike" uniqKey="Hao F" first="Feike" last="Hao">Feike Hao</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/ChloroquineV1/Data/Ncbi/Merge
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000F98 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Ncbi/Merge/biblio.hfd -nk 000F98 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= ChloroquineV1
|flux= Ncbi
|étape= Merge
|type= RBID
|clé= PMC:7058311
|texte= Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Ncbi/Merge/RBID.i -Sk "pubmed:32134989" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Ncbi/Merge/biblio.hfd \
| NlmPubMed2Wicri -a ChloroquineV1
| This area was generated with Dilib version V0.6.33. |  |