An ARF6-Exportin-5 Axis Delivers pre-miRNA Cargo to Tumor Microvesicles.
Identifieur interne : 000851 ( Pmc/Curation ); précédent : 000850; suivant : 000852An ARF6-Exportin-5 Axis Delivers pre-miRNA Cargo to Tumor Microvesicles.
Auteurs : James W. Clancy ; Ye Zhang ; Colin Sheehan ; Crislyn D Ouza-SchoreySource :
- Nature cell biology [ 1465-7392 ] ; 2019.
Abstract
Tumor-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumor cells that are now understood to facilitate communication between the tumor and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-GTP following nuclear export, and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-GTP and GRP1. As such, ARF6 activation increases pre-miRNA cargo contained within TMVs via a process that requires casein kinase 2-mediated phosphorylation of Ran-GAP1. Further, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute 2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.
Url:
DOI: 10.1038/s41556-019-0345-y
PubMed: 31235936
PubMed Central: 6697424
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">An ARF6-Exportin-5 Axis Delivers pre-miRNA Cargo to Tumor Microvesicles.</title><author><name sortKey="Clancy, James W" sort="Clancy, James W" uniqKey="Clancy J" first="James W." last="Clancy">James W. Clancy</name></author><author><name sortKey="Zhang, Ye" sort="Zhang, Ye" uniqKey="Zhang Y" first="Ye" last="Zhang">Ye Zhang</name></author><author><name sortKey="Sheehan, Colin" sort="Sheehan, Colin" uniqKey="Sheehan C" first="Colin" last="Sheehan">Colin Sheehan</name></author><author><name sortKey="D Ouza Schorey, Crislyn" sort="D Ouza Schorey, Crislyn" uniqKey="D Ouza Schorey C" first="Crislyn" last="D Ouza-Schorey">Crislyn D Ouza-Schorey</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31235936</idno><idno type="pmc">6697424</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697424</idno><idno type="RBID">PMC:6697424</idno><idno type="doi">10.1038/s41556-019-0345-y</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000851</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000851</idno><idno type="wicri:Area/Pmc/Curation">000851</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000851</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">An ARF6-Exportin-5 Axis Delivers pre-miRNA Cargo to Tumor Microvesicles.</title><author><name sortKey="Clancy, James W" sort="Clancy, James W" uniqKey="Clancy J" first="James W." last="Clancy">James W. Clancy</name></author><author><name sortKey="Zhang, Ye" sort="Zhang, Ye" uniqKey="Zhang Y" first="Ye" last="Zhang">Ye Zhang</name></author><author><name sortKey="Sheehan, Colin" sort="Sheehan, Colin" uniqKey="Sheehan C" first="Colin" last="Sheehan">Colin Sheehan</name></author><author><name sortKey="D Ouza Schorey, Crislyn" sort="D Ouza Schorey, Crislyn" uniqKey="D Ouza Schorey C" first="Crislyn" last="D Ouza-Schorey">Crislyn D Ouza-Schorey</name></author></analytic><series><title level="j">Nature cell biology</title><idno type="ISSN">1465-7392</idno><idno type="eISSN">1476-4679</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 id="P4">Tumor-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumor cells that are now understood to facilitate communication between the tumor and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-GTP following nuclear export, and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-GTP and GRP1. As such, ARF6 activation increases pre-miRNA cargo contained within TMVs via a process that requires casein kinase 2-mediated phosphorylation of Ran-GAP1. Further, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute 2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Abels, Er" uniqKey="Abels E">ER Abels</name></author><author><name sortKey="Breakefield, Xo" uniqKey="Breakefield X">XO Breakefield</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Desrochers, Lm" uniqKey="Desrochers L">LM Desrochers</name></author><author><name sortKey="Antonyak, Ma" uniqKey="Antonyak M">MA Antonyak</name></author><author><name sortKey="Cerione, Ra" uniqKey="Cerione R">RA Cerione</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tricarico, C" uniqKey="Tricarico C">C Tricarico</name></author><author><name sortKey="Clancy, J" uniqKey="Clancy J">J Clancy</name></author><author><name sortKey="D Ouza Schorey, C" uniqKey="D Ouza Schorey C">C 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<front><journal-meta><journal-id journal-id-type="nlm-journal-id">100890575</journal-id><journal-id journal-id-type="pubmed-jr-id">21417</journal-id><journal-id journal-id-type="nlm-ta">Nat Cell Biol</journal-id><journal-id journal-id-type="iso-abbrev">Nat. Cell Biol.</journal-id><journal-title-group><journal-title>Nature cell biology</journal-title></journal-title-group><issn pub-type="ppub">1465-7392</issn><issn pub-type="epub">1476-4679</issn></journal-meta><article-meta><article-id pub-id-type="pmid">31235936</article-id><article-id pub-id-type="pmc">6697424</article-id><article-id pub-id-type="doi">10.1038/s41556-019-0345-y</article-id><article-id pub-id-type="manuscript">NIHMS1529570</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>An ARF6-Exportin-5 Axis Delivers pre-miRNA Cargo to Tumor Microvesicles.</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Clancy</surname><given-names>James W.</given-names></name><xref ref-type="author-notes" rid="FN2">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Ye</given-names></name><xref ref-type="author-notes" rid="FN2">1</xref></contrib><contrib contrib-type="author"><name><surname>Sheehan</surname><given-names>Colin</given-names></name></contrib><contrib contrib-type="author"><name><surname>D’Souza-Schorey</surname><given-names>Crislyn</given-names></name><xref rid="CR1" ref-type="corresp">*</xref></contrib><aff id="A1">Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46530.</aff></contrib-group><author-notes><corresp id="CR1"><label>*</label>To whom correspondence should be addressed., <email>cdsouzas@nd.edu</email>; Tel: (574) 631-3735</corresp><fn fn-type="con" id="FN1"><p id="P1">Author Contributions:</p><p id="P2">JWC provided conceptual input, designed and performed experiments (<xref rid="F1" ref-type="fig">1a</xref>-<xref rid="F1" ref-type="fig">d</xref>; <xref rid="F2" ref-type="fig">2a</xref>,<xref rid="F2" ref-type="fig">d</xref>,<xref rid="F2" ref-type="fig">e</xref>,<xref rid="F2" ref-type="fig">f</xref>,<xref rid="F2" ref-type="fig">h</xref>; <xref rid="F3" ref-type="fig">3c</xref>,<xref rid="F3" ref-type="fig">e</xref>; <xref rid="F4" ref-type="fig">4</xref>; <xref rid="F5" ref-type="fig">5</xref>; <xref rid="F6" ref-type="fig">6</xref>; <xref rid="SD1" ref-type="supplementary-material">S1a</xref>,e-g; <xref rid="SD1" ref-type="supplementary-material">S2a</xref>, <xref rid="SD1" ref-type="supplementary-material">c</xref>-<xref rid="SD1" ref-type="supplementary-material">g</xref>; <xref rid="SD1" ref-type="supplementary-material">S3b</xref>, <xref rid="SD1" ref-type="supplementary-material">d</xref>-<xref rid="SD1" ref-type="supplementary-material">g</xref>; <xref rid="SD1" ref-type="supplementary-material">S4</xref>; <xref rid="SD1" ref-type="supplementary-material">S5</xref>; <xref rid="SD1" ref-type="supplementary-material">S6a</xref>-<xref rid="SD1" ref-type="supplementary-material">c</xref>; S7a-c, e-j-k), analyzed the data, proposed the model, assembled the figures and wrote the manuscript; YZ provided conceptual input, designed and performed experiments (<xref rid="F1" ref-type="fig">1e</xref>-<xref rid="F1" ref-type="fig">l</xref>; <xref rid="F2" ref-type="fig">2a</xref>-<xref rid="F2" ref-type="fig">e</xref>,<xref rid="F2" ref-type="fig">i</xref>; <xref rid="F3" ref-type="fig">3a</xref>-<xref rid="F3" ref-type="fig">e</xref>; <xref rid="F4" ref-type="fig">4a</xref>, <xref rid="F5" ref-type="fig">5</xref>; <xref rid="F6" ref-type="fig">6f</xref>; <xref rid="SD1" ref-type="supplementary-material">S1b</xref>,<xref rid="SD1" ref-type="supplementary-material">c</xref>; <xref rid="SD1" ref-type="supplementary-material">S2a</xref>-<xref rid="SD1" ref-type="supplementary-material">c</xref>; <xref rid="SD1" ref-type="supplementary-material">S3a</xref>,<xref rid="SD1" ref-type="supplementary-material">b</xref>,<xref rid="SD1" ref-type="supplementary-material">j</xref>; <xref rid="SD1" ref-type="supplementary-material">S4a</xref>; <xref rid="SD1" ref-type="supplementary-material">S6d</xref>, <xref rid="SD1" ref-type="supplementary-material">j</xref>), analyzed the data and contributed to writing of the manuscript; CS designed and performed experiments (<xref rid="SD1" ref-type="supplementary-material">S1b</xref>; <xref rid="SD1" ref-type="supplementary-material">S6j</xref>) and assisted with experiments; CD-S provided conceptual input, contributed to experimental design, analyzed the data, wrote the manuscript and was responsible for overall project administration.</p></fn><fn id="FN2"><label>1</label><p id="P3">Co-First Authors</p></fn></author-notes><pub-date pub-type="nihms-submitted"><day>6</day><month>6</month><year>2019</year></pub-date><pub-date pub-type="epub"><day>24</day><month>6</month><year>2019</year></pub-date><pub-date pub-type="ppub"><month>7</month><year>2019</year></pub-date><pub-date pub-type="pmc-release"><day>24</day><month>12</month><year>2019</year></pub-date><volume>21</volume><issue>7</issue><fpage>856</fpage><lpage>866</lpage><pmc-comment>elocation-id from pubmed: 10.1038/s41556-019-0345-y</pmc-comment>
<permissions><license><license-p>Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:<uri xlink:type="simple" xlink:href="http://www.nature.com/authors/editorial_policies/license.html#terms">http://www.nature.com/authors/editorial_policies/license.html#terms</uri></license-p></license></permissions><abstract id="ABS1"><p id="P4">Tumor-derived microvesicles (TMVs) comprise a class of extracellular vesicles released from tumor cells that are now understood to facilitate communication between the tumor and the surrounding microenvironment. Despite their significance, the regulatory mechanisms governing the trafficking of bioactive cargos to TMVs at the cell surface remain poorly defined. Here we describe a molecular pathway for the delivery of microRNA (miRNA) cargo to nascent TMVs involving the dissociation of a pre-miRNA/Exportin-5 complex from Ran-GTP following nuclear export, and its subsequent transfer to a cytoplasmic shuttle comprised of ARF6-GTP and GRP1. As such, ARF6 activation increases pre-miRNA cargo contained within TMVs via a process that requires casein kinase 2-mediated phosphorylation of Ran-GAP1. Further, TMVs were found to contain pre-miRNA processing machinery including Dicer and Argonaute 2, which allow for cell-free pre-miRNA processing within shed vesicles. These findings offer cellular targets to block the loading and processing of pre-miRNAs within TMVs.</p></abstract></article-meta></front><floats-group><fig id="F1" orientation="portrait" position="float"><label>Figure 1:</label><caption><title>TMVs are a distinct class of extracellular vesicles and contain pre-miRNA cargo.</title><p id="P47">Whole LOX melanoma cells (<bold>a</bold>), isolated LOX TMVs (<bold>b</bold>), or isolated LOX exosomes (<bold>c</bold>) were analyzed by scanning electron microscopy. Representative images of each population shown. Images are representative of N=3 biological and n=2 technical replicates. <bold>d.</bold> 20 μg of total protein isolate from whole cells (LOX Cell) or isolated TMVs (LOX TMV) was separated by SDS-PAGE and protein cargo determined by western blotting. Blots are representative of N=3 independent biological experiments. <bold>e.</bold> Agilent bioanalyzer analysis of TMV RNA content using the RNA 6000 Nano kit shows cargo corresponding in size to pre-miRNA (green bar). <bold>f.</bold> Higher resolution analysis of TMV small RNA using the Bioanalyzer Small RNA 6–150 nt Analysis kit shows peak corresponding to mature miRNA (green bar). Representative images from N=3 independent biological samples shown. <bold>g.</bold> Bowtie analysis of small RNA content isolated from TMVs released from invasive tumor cell lines of melanoma (LOX), prostate (PC-3), and breast (MDA-MB-231) origin. For each cell type, N=3 independent biological samples. <bold>h.</bold> ARF6 activity was measured using an MT2 ARF6-GTP specific pulldown assay as described in methods. Data presented is representative of N=4 independent biological experiments-. ARF6 <bold>i.</bold> Analysis of small RNA content isolated from parental melanoma cells or those expressing constitutively active ARF6-GTP showed no difference in the quantity of detectable RNA with ARF6 activation. <bold>j.</bold> Sequencing analysis revealed a significant increase in the total read hits corresponding to miRNA upon expression of ARF6-Q67L. <bold>k.</bold> qRT-PCR analysis confirms the increase in pre-miRNA cargo content in TMVs released by tumor cells of melanoma (LOX), ovarian (OvCar3), and breast (MDA-MB-231) origins. <bold>l.</bold> qRT-PCR analysis of total cell pre-miRNA levels with expression of ARF6-Q67L. Data presented as mean±standard deviation. For panels <bold>b-e</bold>, statistical analysis was based on measurements obtained for 3 biological repeats (N=3). P-values determined by unpaired, two-tailed t-test between control and treatment reactions for each independent experimental condition. P-values <0.05 were considered significant. Unprocessed blot images shown in <xref rid="SD1" ref-type="supplementary-material">Supplemental Image 7</xref>. Statistical Source in <xref rid="SD2" ref-type="supplementary-material">Supplementary Table 1</xref>.</p></caption><graphic xlink:href="nihms-1529570-f0001"></graphic></fig><fig id="F2" orientation="portrait" position="float"><label>Figure 2:</label><caption><title>ARF6 interacts with the pre-miRNA transport protein Exportin-5.</title><p id="P48"><bold>a.</bold> PRISM prediction was used to model the predicted interaction between Exportin-5 and ARF6-GTPγS. <bold>b.</bold> Predicted binding energies between the Exportin-5 (3a6p) and ARF6-GTPγS (2j5x), or ARF6-GDP (1eos). <bold>c.</bold> Exportin-5 co-immunoprecipitates with ARF6 confirming the interaction predicted using PRISM. 200 μg of total protein was subjected to immunoprecipitation as described in methods. Antibody bound protein was precipitated using Protein-G conjugated Dynabeads before being resolved by SDS-PAGE and examined by western blotting. N=3 biologically independent experiments. <bold>d.</bold> Exportin-5 preferentially binds active, GTP-bound ARF6 <italic>in vitro</italic>. Recombinant GST-wt-ARF6 conjugated beads were incubated with lysates in the presence of 100 μM GTP-γ-S, 1 mM GDP, or vehicle control for 60 min at 37˚C. Bound proteins were precipitated and separated by SDS-PAGE for western blotting to examine relative amounts of co-precipitating Exportin-5. Data presented as mean±SD (N=4 biologically independent experiments). p-values determined by one-way ANOVA with Sidak’s correction for multiple comparisons. p-values <0.05 were considered significant. <bold>e.</bold> Dominant negative ARF6-T27N inhibits Exportin-5 co-precipitation. 200 μg of total protein from cells transiently expressing wt-ARF6 or ARF6-T27N was subjected to immunoprecipitation as described in methods. Immunoprecipitated protein was resolved by SDS-PAGE and examined by western blotting as indicated (N=3 biologically independent experiments). <bold>f.</bold> Immunofluorescent analysis of endogenous Exportin-5 in invasive melanoma cells reveals intracellular distribution divided between nuclear and cytoplasmic pools. Scale bar = 15 μm. <bold>g.</bold> Higher magnification analysis of Exportin-5 localization indicating the inclusion of Exportin-5 in nascent TMVs at the cell periphery (arrows). Scale bar = 15 μm. Panels <bold>f-g</bold> represent N=5 biologically independent experiments) <bold>h.</bold> Endogenous Exportin-5 and ARF6 co-localize in nascent TMVs at the cell periphery (arrows). Scale bar = 25 μm (N=3 biologically independent experiments) <bold>i.</bold> Western blot analysis of cargo content from TMVs isolated from multiple invasive cell lines confirms the inclusion of Exportin-5 as TMV cargo. (N=3 biologically independent experiments). Unprocessed blot images shown in <xref rid="SD1" ref-type="supplementary-material">Supplemental Image 7</xref>. Statistical Source in <xref rid="SD2" ref-type="supplementary-material">Supplementary Table 1</xref>.</p></caption><graphic xlink:href="nihms-1529570-f0002"></graphic></fig><fig id="F3" orientation="portrait" position="float"><label>Figure 3:</label><caption><title>pre-miRNA processing machinery is contained in shed TMVs.</title><p id="P49"><bold>a.</bold> TMVs from multiple invasive tumor cell lines were analyzed for the inclusion of pre-miRNA processing proteins Dicer and Argonaute-2 by western blotting. N=4 biologically independent samples. <bold>b.</bold> Western blot analysis of lysate generated from equal number of (1×10<sup>8</sup>) TMVs released by parental melanoma cells compared to those expressing constitutively active ARF6 reveals an enrichment of Exportin-5 content within TMVs when ARF6 is activated. Data presented as mean±SD (N=3 biologically independent experiments). P-value determined by unpaired two-tailed t-test. P-value <0.05 was considered significant. <bold>c.</bold> Western blot analysis of lysate generated from equal number of (1×10<sup>8</sup>) TMVs released by parental melanoma cells compared to those transfected with fast-cycling ARF6-T157N confirms an enrichment of Exportin-5 content within TMVs when ARF6 is activated. Data presented as mean±SD (N=3 biologically independent experiments). P-value determined by unpaired two-tailed t-test. p-value <0.05 was considered significant. <bold>d.</bold> RNA extracted from equal numbers (5×10<sup>6</sup>) of isolated TMVs maintained in cell-free conditions at 37˚C for the times indicated was analyzed by qRT-PCR. The relative amounts of pre-miR21 and mature miR-21 were measured as described in methods. Data presented as mean±SD from N=3 biologically independent experiments. <bold>e.</bold> TMVs were isolated from invasive melanoma cells and endogenous Exportin-5 precipitated from 200 μg of input TMV protein. Co-precipitating RNA was examined by RT-PCR, and co-precipitating proteins examined by western blotting. Representative data from N=3 biologically independent samples shown. Unprocessed blot images shown in <xref rid="SD1" ref-type="supplementary-material">Supplemental Image 7</xref>. Statistical Source in <xref rid="SD2" ref-type="supplementary-material">Supplementary Table 1</xref>.</p></caption><graphic xlink:href="nihms-1529570-f0003"></graphic></fig><fig id="F4" orientation="portrait" position="float"><label>Figure 4:</label><caption><title>Casein Kinase 2 activity is needed for Exportin-5 trafficking.</title><p id="P50"><bold>a.</bold> Western blot analysis lysates generated from equal numbers of control or TBB treated cells (5×10<sup>5</sup>) or TMVs (1×10<sup>8</sup>). Data presented as mean±SD from N=3 biologically independent experiments. P-value determined by unpaired two-tailed t-test. P-value <0.05 was considered significant. <bold>b.</bold> RNA isolated from TMVs with or without TBB treatment was analyzed using qRT-PCR. CK2 inhibition results in a decrease in pre-miRNA contained within TMVs. Data presented as mean±SD for N=3 biologically independent experiments. P-values determined by unpaired, two-tailed t-test between control and treatment reactions for each independent pre-miRNA. P-values <0.05 were considered significant. <bold>c.</bold> Intracellular distribution of Exportin-5 was examined by immunofluorescence in cells treated with TBB or DMSO vehicle control. Scale bars = 15 μm. Representative images from N=3 biologically independent experiments shown. <bold>d.</bold> Heat map visualization of Exportin-5 channel described in <bold>c</bold>. <bold>e.</bold> LOX cells expressing constitutively active ARF6-Q67L were treated with TBB to block CK2 activity. The localization of Exportin-5 was then examined by immunofluorescent microscopy. Representative images (N=3 biologically independent experiments) shown. <bold>f.</bold> Heat map visualization of Exportin-5 channel described in <bold>e</bold>. <bold>g.</bold> Equal numbers of TMVs (1×10<sup>8</sup>) were isolated from LOX<sup>ARF6-GTP</sup> cells and subjected to western blot analysis to examine the levels of Exportin-5 contained as TMV cargo. Data presented as mean±SD (N=3 biologically independent experiments). p-value determined by unpaired two-tailed t-test. p-value <0.05 was considered significant. <bold>h.</bold> Melanoma cells expressing GFP-RanGAP were subjected to GFP-Trap and western blotting to examine changes in phosphoserine levels upon treatment with TBB. Representative data from N=3 biologically independent experiments shown. <bold>i.</bold> ARF6 was immunoprecipitated from cells with CK2 inhibition. The amount of Exportin-5 and Ran that co-precipitated with the GTPase was then examined by western blotting. Representative images from N=3 biologically independent experiments shown <bold>j.</bold> Exportin-5 was precipitated from cells treated with TBB and the levels of Ran and ARF6 which co-precipitated were subsequently examined by western blotting. Data is representative of N=3 biologically independent repeats. Unprocessed blot images shown in <xref rid="SD1" ref-type="supplementary-material">Supplemental Image 7</xref>. Statistical Source in <xref rid="SD2" ref-type="supplementary-material">Supplementary Table 1</xref>.</p></caption><graphic xlink:href="nihms-1529570-f0004"></graphic></fig><fig id="F5" orientation="portrait" position="float"><label>Figure 5:</label><caption><title>GRP1 scaffolding function facilitates Exportin-5 trafficking to TMVs.</title><p id="P51"><bold>a.</bold> Exportin-5 localization was examined by immunofluorescence in the presence of SecinH3. Scale bar = 15 μm. Representative images (N=5 biologically independent experiments shown. <bold>b.</bold> Exportin-5 western blot from equal numbers of TMVs from control or SecinH3 treated cells. Data presented as mean±SD for N=4 biologically independent experiments. P-value calculated using unpaired two-tailed t-test. <bold>c.</bold> Melanoma cells were co-treated with SecinH3 and chloroquine, and the intracellular distribution of Exportin-5 examined by confocal microscopy. Scale bar = 15 μm. Images representative of N=3 biologically independent experiments. <bold>d.</bold> Predicted interaction between Exportin-5 and ARF6 with GRP1 (4kax) was modeled, highlighting the energetically favorable binding arrangements listed in <bold>e</bold>. <bold>f.</bold> Exportin-5 co-immunoprecipitation from melanoma cells was analyzed by western blot to confirm the inclusion of GRP1 in complex with Exportin-5 and ARF6. Blots represent N=3 biologically independent experiments. <bold>g.</bold> Endogenous GRP1 was depleted from melanoma cells prior to immunoprecipitation of HA-ARF6. Co-precipitating proteins were separated by SDS-PAGE and analyzed by western blotting as indicated. Representative blots (N=3 biologically independent experiments) shown. <bold>h.</bold> Levels of Exportin-5, Dicer, and Argonaute-2 TMV cargo were analyzed by western blotting following isolation from cells depleted of endogenous GRP1. Data presented as mean±SD (N=5 biologically independent samples). P-value determined by unpaired two-tailed t-test. <bold>i, j.</bold> TMV pre-miRNA content was isolated from cells expressing 2 independent shRNA hairpins against GRP1. Isolated RNA was then analyzed by qRT-PCR. For each condition Data presented as mean±SD from 3 biologically independent experiments. P-values determined by unpaired, two-tailed t-test between control and treatment reactions for each independent pre-miRNA amplification reaction. <bold>k.</bold> Equal numbers of TMVs (1×10<sup>8</sup>) were isolated from control or GRP1-shRNA treated LOX<sup>ARF6-Q67L</sup> cells and lysates analyzed by western blotting as indicated. <bold>l.</bold> ARF6 was immunoprecipitated from LOX<sup>ARF6-Q67L</sup> cells with or without GRP1-shRNA. Co-precipitating proteins were examined by western blot as indicated. For panels <bold>k-l</bold> representative blots (N=3 biologically independent experiments) shown. In all panels, p-values <0.05 were considered significant. Unprocessed blot images shown in <xref rid="SD1" ref-type="supplementary-material">Supplemental Image 7</xref>. Statistical Source in <xref rid="SD2" ref-type="supplementary-material">Supplementary Table 1</xref>.</p></caption><graphic xlink:href="nihms-1529570-f0005"></graphic></fig><fig id="F6" orientation="portrait" position="float"><label>Figure 6:</label><caption><title>TMVs transfer functional miRNAs to recipient cells.</title><p id="P52"><bold>a.</bold> Experimental scheme to knockdown endogenous miR-21 using HDR and CRISPR/Cas-9. <bold>b.</bold> 5,000 control; pL552-miR-21 transfected; or pL552-miR-21 and PX458-miR-21 dual transfected LOX cells were treated with 400 ng/mL puromycin (time t=0), and cell death measured. Data presented as mean±SD from 3 biologically independent samples. <bold>c.</bold> qRT-PCR of miR-21 in knockdown colonies. Data presented as mean±SD from 3 biologically independent samples. <bold>d.</bold> Western blotting of TIMP3 using 20 μg of lysate from control or miR-21 knockdown cells. Representative blots (N=3 biologically independent experiments) shown. <bold>e.</bold> Fibroblasts were incubated with SYTO RNAselect-stained TMVs for 24 hours before processing for confocal microscopy. Scale bar = 20 μm. Representative images (N=4 biologically independent experiments) shown. <bold>f.</bold> LOX or LOX<sup>miR−21KO</sup> cells were transfected with miRNA sponges as indicated. Transfected cells were incubated with Isolated TMVs for 10 hours before being lysed and levels of dEGFP protein examined by western blot. Representative images shown (N=3 biologically independent experiments). <bold>g.</bold> LOX or LOX<sup>miR−21KO</sup> cells were transfected with miRNA sponges and incubated with isolated TMVs for 10 hours before cells were lysed and dEGFP mRNA quantified by qRT-PCR. Mean±SD for N=3 biologically independent experiments shown. No statistically significant relationships were found. <bold>h.</bold> BJ fibroblasts were incubated with purified LOX TMVs for 48 hours and alpha smooth muscle actin levels measured by western blotting using 20 μg of cell lysates. Data presented as mean±SD (N=3 biologically independent samples). <bold>i.</bold> SMAD-7 levels were measured using 20 μg of BJ fibroblast cell lysate following treatment with isolated TMVs. Mean±SD (N=4 biologically independent experiments) shown. <bold>j.</bold> SMAD-7 mRNA levels in TMV-treated BJ fibroblasts were measured by qRT-PCR as described in methods. Data presented as mean±SD (N=4 biologically independent experiments). p-values determined by multiple unpaired two-tailed t-tests with Bonferroni’s correction (<bold>c, d, h</bold>); two-way ANOVA (<bold>g</bold>); or one-way ANOVA with Bonferroni’s correction (<bold>i, j</bold>).For all panels, p-values <0.05 were considered significant. Unprocessed blot images shown in <xref rid="SD1" ref-type="supplementary-material">Supplemental Image 7</xref>. Statistical Source in <xref rid="SD2" ref-type="supplementary-material">Supplementary Table 1</xref>.</p></caption><graphic xlink:href="nihms-1529570-f0006"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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