Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells
Identifieur interne : 000B41 ( Ncbi/Merge ); précédent : 000B40; suivant : 000B42Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells
Auteurs : Arnold Sipos [États-Unis] ; Kwang-Jin Kim [États-Unis] ; Constantinos Sioutas ; Edward D. Crandall [États-Unis]Source :
- International Journal of Molecular Sciences [ 1422-0067 ] ; 2019.
Abstract
Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.
Url:
DOI: 10.3390/ijms20215253
PubMed: 31652767
PubMed Central: 6861930
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type="state">Californie</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="af7-ijms-20-05253">Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9092, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9092</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="af8-ijms-20-05253">Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-1211, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Mork Family Department of Chemical Engineering and Materials Science, 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USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Kim, Kwang Jin" sort="Kim, Kwang Jin" uniqKey="Kim K" first="Kwang-Jin" last="Kim">Kwang-Jin Kim</name><affiliation><nlm:aff id="af1-ijms-20-05253">Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906, USA;<email>kjkim@usc.edu</email> (K.-J.K.);<email>edward.crandall@med.usc.edu</email> (E.D.C.)</nlm:aff></affiliation><affiliation wicri:level="4"><nlm:aff id="af2-ijms-20-05253">Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="af3-ijms-20-05253">Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089-9037, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089-9037</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="af4-ijms-20-05253">Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="af5-ijms-20-05253">Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-1111, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-1111</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation></author><author><name sortKey="Sioutas, Constantinos" sort="Sioutas, Constantinos" uniqKey="Sioutas C" first="Constantinos" last="Sioutas">Constantinos Sioutas</name><affiliation><nlm:aff id="af6-ijms-20-05253">Sonny Astani Department of Civil and Environmental Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-2531, USA;<email>sioutas@usc.edu</email></nlm:aff></affiliation></author><author><name sortKey="Crandall, Edward D" sort="Crandall, Edward D" uniqKey="Crandall E" first="Edward D." last="Crandall">Edward D. Crandall</name><affiliation><nlm:aff id="af1-ijms-20-05253">Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906, USA;<email>kjkim@usc.edu</email> (K.-J.K.);<email>edward.crandall@med.usc.edu</email> (E.D.C.)</nlm:aff></affiliation><affiliation wicri:level="4"><nlm:aff id="af2-ijms-20-05253">Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="af7-ijms-20-05253">Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9092, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9092</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="af8-ijms-20-05253">Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-1211, USA</nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea>Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-1211</wicri:regionArea><orgName type="university">Université de Californie du Sud</orgName><placeName><settlement type="city">Los Angeles</settlement><region type="state">Californie</region></placeName></affiliation></author></analytic><series><title level="j">International Journal of Molecular Sciences</title><idno type="eISSN">1422-0067</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca<sup>2+</sup>]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca<sup>2+</sup>]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Buzea, C" uniqKey="Buzea C">C. Buzea</name></author><author><name sortKey="Pacheco, I I" uniqKey="Pacheco I">I.I. Pacheco</name></author><author><name sortKey="Robbie, K" uniqKey="Robbie K">K. Robbie</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Khan, M I" uniqKey="Khan M">M.I. Khan</name></author><author><name sortKey="Mohammad, A" uniqKey="Mohammad A">A. Mohammad</name></author><author><name sortKey="Patil, G" uniqKey="Patil G">G. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Mol Sci</journal-id><journal-id journal-id-type="iso-abbrev">Int J Mol Sci</journal-id><journal-id journal-id-type="publisher-id">ijms</journal-id><journal-title-group><journal-title>International Journal of Molecular Sciences</journal-title></journal-title-group><issn pub-type="epub">1422-0067</issn><publisher><publisher-name>MDPI</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31652767</article-id><article-id pub-id-type="pmc">6861930</article-id><article-id pub-id-type="doi">10.3390/ijms20215253</article-id><article-id pub-id-type="publisher-id">ijms-20-05253</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Sipos</surname><given-names>Arnold</given-names></name><xref ref-type="aff" rid="af1-ijms-20-05253">1</xref><xref ref-type="aff" rid="af2-ijms-20-05253">2</xref><xref rid="c1-ijms-20-05253" ref-type="corresp">*</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0001-7547-1681</contrib-id><name><surname>Kim</surname><given-names>Kwang-Jin</given-names></name><xref ref-type="aff" rid="af1-ijms-20-05253">1</xref><xref ref-type="aff" rid="af2-ijms-20-05253">2</xref><xref ref-type="aff" rid="af3-ijms-20-05253">3</xref><xref ref-type="aff" rid="af4-ijms-20-05253">4</xref><xref ref-type="aff" rid="af5-ijms-20-05253">5</xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="true">https://orcid.org/0000-0001-5146-0857</contrib-id><name><surname>Sioutas</surname><given-names>Constantinos</given-names></name><xref ref-type="aff" rid="af6-ijms-20-05253">6</xref></contrib><contrib contrib-type="author"><name><surname>Crandall</surname><given-names>Edward D.</given-names></name><xref ref-type="aff" rid="af1-ijms-20-05253">1</xref><xref ref-type="aff" rid="af2-ijms-20-05253">2</xref><xref ref-type="aff" rid="af7-ijms-20-05253">7</xref><xref ref-type="aff" rid="af8-ijms-20-05253">8</xref></contrib></contrib-group><aff id="af1-ijms-20-05253"><label>1</label>Will Rogers Institute Pulmonary Research Center and Hastings Center for Pulmonary Research, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906, USA;<email>kjkim@usc.edu</email> (K.-J.K.);<email>edward.crandall@med.usc.edu</email> (E.D.C.)</aff><aff id="af2-ijms-20-05253"><label>2</label>Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-0906, USA</aff><aff id="af3-ijms-20-05253"><label>3</label>Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089-9037, USA</aff><aff id="af4-ijms-20-05253"><label>4</label>Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121, USA</aff><aff id="af5-ijms-20-05253"><label>5</label>Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-1111, USA</aff><aff id="af6-ijms-20-05253"><label>6</label>Sonny Astani Department of Civil and Environmental Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-2531, USA;<email>sioutas@usc.edu</email></aff><aff id="af7-ijms-20-05253"><label>7</label>Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033-9092, USA</aff><aff id="af8-ijms-20-05253"><label>8</label>Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089-1211, USA</aff><author-notes><corresp id="c1-ijms-20-05253"><label>*</label>Correspondence: <email>asipos@usc.edu</email></corresp></author-notes><pub-date pub-type="epub"><day>23</day><month>10</month><year>2019</year></pub-date><pub-date pub-type="collection"><month>11</month><year>2019</year></pub-date><volume>20</volume><issue>21</issue><elocation-id>5253</elocation-id><history><date date-type="received"><day>29</day><month>9</month><year>2019</year></date><date date-type="accepted"><day>21</day><month>10</month><year>2019</year></date></history><permissions><copyright-statement>© 2019 by the authors.</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access"><license-p>Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>).</license-p></license></permissions><abstract><p>Background: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca<sup>2+</sup>]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. Methods: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. Results: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca<sup>2+</sup>]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. Conclusions: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.</p></abstract><kwd-group><kwd>autophagy</kwd><kwd>lysosome</kwd><kwd>lysosomal membrane permeability</kwd><kwd>mitochondria</kwd><kwd>pneumocyte</kwd></kwd-group></article-meta></front><floats-group><fig id="ijms-20-05253-f001" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Intracellular accumulation of polystyrene nanoparticles (PNP) in A549 cells. Apical exposure of A549 cells to PNP at 80 μg/mL for 24 h led to accumulation of PNP (red) in intracellular vesicles, while diffuse distribution of PNP in cytosol was also seen. Plasma membranes of A549 cells were labeled by Dylight 488-conjugated tomato lectin (green). Scale bar is 10 μm.</p></caption><graphic xlink:href="ijms-20-05253-g001"></graphic></fig><fig id="ijms-20-05253-f002" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Relative changes in intracellular PNP content in A549 cells in the presence or absence (control) of endocytosis inhibitors after 24 h of apical PNP exposure. Monodansylcadaverine (MDC, an inhibitor of clathrin-mediated endocytosis) failed to decrease intracellular PNP content, whereas cytochalasin D (CCD, an inhibitor of macropinocytosis) and nocodazole (an inhibitor of microtubule polymerization) decreased intracellular PNP content by 60% and 17%, respectively. * <italic>p</italic> < 0.05 compared to control.</p></caption><graphic xlink:href="ijms-20-05253-g002"></graphic></fig><fig id="ijms-20-05253-f003" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Colocalization of early endosome marker Rab5a-GFP with PNP in A549 cells. A549 cells were transduced for 2 h with an early endosome marker (Rab5a-GFP, green) and apically exposed thereafter to PNP (red) for 24 h. Colocalization (arrowheads, yellow) of PNP with Rab5a-GFP-positive vesicles was observed in some of the vesicles. Contours of cells were added (dotted lines) on the basis of the cell plasma membrane marker Dylight 405-conjugated tomato lectin (blue). Images are representative of 4–5 observations. Scale bar is 10 μm.</p></caption><graphic xlink:href="ijms-20-05253-g003"></graphic></fig><fig id="ijms-20-05253-f004" orientation="portrait" position="float"><label>Figure 4</label><caption><p>PNP egress from A549 cells. (<bold>a</bold>) A549 cells were apically exposed to PNP for 12 h, followed by washing with fresh culture fluid and assessing intracellular PNP content at designated time points for up to 24 h thereafter. When 10 μM ATP was applied apically to A549 cells at time zero and remained present throughout the entire experiment, no difference in PNP egress kinetics between control (no stimulation) and ATP-treated A549 cells during egress was observed. <italic>n</italic> = 4–6 for each time point. (<bold>b</bold>) Representative recording of oscillations in intracellular [Ca<sup>2+</sup>] detected upon 2.5 min presence of 10 μM ATP in the apical bathing fluid of A549 cells. Different colors represent intracellular [Ca<sup>2+</sup>] observed in two different A549 cells.</p></caption><graphic xlink:href="ijms-20-05253-g004"></graphic></fig><fig id="ijms-20-05253-f005" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Apical nanoparticle (NP) exposure induced activation of autophagy in A549 cells. A549 cells were preincubated with chloroquine (40 μM, 30 min) and exposed thereafter to NP (PNP or ambient air pollution particles (PM0.2)) for 24 h in the continued presence of chloroquine, followed by assessment of LC3 expression by immunolabeling. LC3 expression (red) was detected in NP-exposed A549 cells. No or very low level of LC3 expression was found in control cells not exposed to NP. Plasma membranes of A549 cells were labeled by Dylight 488-conjugated tomato lectin (green), whereas nuclei were labeled by Hoechst 33342 (blue). Images are representative of 4–5 observations. Scale bars are 25 μm.</p></caption><graphic xlink:href="ijms-20-05253-g005"></graphic></fig><fig id="ijms-20-05253-f006" orientation="portrait" position="float"><label>Figure 6</label><caption><p>Colocalization of PNP with LC3-GFP in A549 cells. Following transduction of A549 cells with the autophagosome marker LC3-GFP construct for 2 h, cells were preincubated with chloroquine (40 μM) and apically exposed thereafter to PNP for 24 h in the continued presence of chloroquine. Colocalization of PNP (red) with LC3-GFP-positive vesicles (green) was observed. Contour of cell was added (dotted line) on the basis of the cell plasma membrane marker Dylight 405-conjugated tomato lectin (blue). Images are representative of 4–5 observations. Scale bar is 10 μm.</p></caption><graphic xlink:href="ijms-20-05253-g006"></graphic></fig><fig id="ijms-20-05253-f007" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Effects of autophagy inhibitors on intracellular PNP content in A549 cells at 24 h post-exposure to PNP. Intracellular PNP content in A549 cells was reduced by 38% and 64%, respectively, when autophagosome formation was inhibited with 3-methyladenine (3-MA) or autophagosome-lysosome fusion was inhibited with bafilomycin. Data are normalized to control. * <italic>p</italic> < 0.05 compared to control.</p></caption><graphic xlink:href="ijms-20-05253-g007"></graphic></fig><fig id="ijms-20-05253-f008" orientation="portrait" position="float"><label>Figure 8</label><caption><p>Colocalization of PNP with lysosomes in A549 cells. At 24 h post exposure to PNP, lysosomes in A549 cells were labeled with Lysotracker Green (green). Colocalization (yellow; arrowheads) can be seen between PNP (red) and Lysotracker Green in some of the vesicles in the perinuclear area. Plasma membrane of A549 cells was labeled by Dylight 405-conjugated tomato lectin (blue or shown with dotted line). Images are representative of 4–5 observations. Scale bar is 10 μm.</p></caption><graphic xlink:href="ijms-20-05253-g008"></graphic></fig><fig id="ijms-20-05253-f009" orientation="portrait" position="float"><label>Figure 9</label><caption><p>Detection of increased lysosomal membrane permeability (LMP) using acridine orange (AO) in A549 cells. (<bold>a</bold>) Increased LMP was detected when AO (green; ex/em: 488/500–525 nm) intensity in lysosomes was decreased along with increased AO intensity in cytoplasm and nucleus. In “no NP” panels, A549 cells showed virtually no detectable nuclear AO signal. A549 cells pretreated with ciprofloxacin (CPX, 150 μM for 24 h, without NP exposure) as positive control exhibited AO accumulation in both nucleus (arrow) and cytoplasm (arrowhead). NP (PNP or PM0.2) exposure also led to AO accumulation in cytoplasm and nucleus. Contours of cells were added (dotted line) on the basis of the cell plasma membrane marker Dylight 405-conjugated tomato lectin (blue). Scale bars are 20 μm. (<bold>b</bold>) When A549 cells were apically exposed to either PNP or PM0.2 for 24 h, increased LMP index (AO green fluorescence intensity in cytoplasm and nucleus/total cellular AO green fluorescence intensity) was found to be similar to that of positive control (CPX). <italic>n</italic> = 4–6. * <italic>p</italic> < 0.05 compared to control.</p></caption><graphic xlink:href="ijms-20-05253-g009"></graphic></fig><fig id="ijms-20-05253-f010" orientation="portrait" position="float"><label>Figure 10</label><caption><p>Effect of apical NP exposure for 24 h on mitochondrial membrane potential in A549 cells. Mitochondrial membrane potentials were normalized to the corresponding controls (i.e., not exposed to either NP or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)). Mitochondrial membrane potential was nearly abolished in the presence of FCCP (1 μM, positive control), whereas it did not decrease following 24 h of apical exposure to PNP or PM0.2. * <italic>p</italic> < 0.05 compared to control.</p></caption><graphic xlink:href="ijms-20-05253-g010"></graphic></fig><fig id="ijms-20-05253-f011" orientation="portrait" position="float"><label>Figure 11</label><caption><p>Absence of mitophagy in NP-exposed A549 cells. Mitophagy is detected on the basis of an increase in the fluorescent signal of the Mtphagy dye (in comparison to that of control; red) and colocalization of the Mtphagy dye with lysosomal indicator (LI; green) dye. In control (no exposure to PNP or PM0.2), the Mtphagy dye had weak fluorescence and minimal colocalization with lysosomes. After FCCP exposure (positive control), the Mtphagy dye exhibited strong fluorescence with extensive colocalization with the LI lysosomal marker dye. Exposure of A549 cells to either PNP or PM0.2 yielded weak fluorescence with the Mtphagy dye and showed minimal lysosomal colocalization of the Mtphagy dye, consistent with a low level (or relative absence) of mitophagy. Contours of cells were added (dotted line) on the basis of the cell plasma membrane marker Dylight 405-conjugated tomato lectin. Scale bars (25 μm) are shown in right panels only.</p></caption><graphic xlink:href="ijms-20-05253-g011"></graphic></fig></floats-group></pmc><affiliations><list><country><li>États-Unis</li></country><region><li>Californie</li></region><settlement><li>Los Angeles</li></settlement><orgName><li>Université de Californie du Sud</li></orgName></list><tree><noCountry><name sortKey="Sioutas, Constantinos" sort="Sioutas, Constantinos" uniqKey="Sioutas C" first="Constantinos" last="Sioutas">Constantinos Sioutas</name></noCountry><country name="États-Unis"><region name="Californie"><name sortKey="Sipos, Arnold" sort="Sipos, Arnold" uniqKey="Sipos A" first="Arnold" last="Sipos">Arnold Sipos</name></region><name sortKey="Crandall, Edward D" sort="Crandall, Edward D" uniqKey="Crandall E" first="Edward D." last="Crandall">Edward D. Crandall</name><name sortKey="Crandall, Edward D" sort="Crandall, Edward D" uniqKey="Crandall E" first="Edward D." last="Crandall">Edward D. Crandall</name><name sortKey="Crandall, Edward D" sort="Crandall, Edward D" uniqKey="Crandall E" first="Edward D." last="Crandall">Edward D. Crandall</name><name sortKey="Kim, Kwang Jin" sort="Kim, Kwang Jin" uniqKey="Kim K" first="Kwang-Jin" last="Kim">Kwang-Jin Kim</name><name sortKey="Kim, Kwang Jin" sort="Kim, Kwang Jin" uniqKey="Kim K" first="Kwang-Jin" last="Kim">Kwang-Jin Kim</name><name sortKey="Kim, Kwang Jin" sort="Kim, Kwang Jin" uniqKey="Kim K" first="Kwang-Jin" last="Kim">Kwang-Jin Kim</name><name sortKey="Kim, Kwang Jin" sort="Kim, Kwang Jin" uniqKey="Kim K" first="Kwang-Jin" last="Kim">Kwang-Jin Kim</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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