212d589bf10d3fe0d7b72d517d5bc26c pmc_27882925.pdf 324b959bcc76ba49678a482264e62ee90db6512c pmc_27882925.pdf 9f511fc3200e254500c37379caab863389f727f150f64e599553808da0c25a42 pmc_27882925.pdf Title: untitled Creator: Arbortext Advanced Print Publisher 9.0.226/W Producer: Acrobat Distiller 8.1.0 (Windows) CreationDate: Thu Nov 17 10:01:07 2016 CET ModDate: Thu Nov 17 10:01:07 2016 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 11 Encrypted: no Page size: 595.276 x 782.362 pts Page rot: 0 File size: 3268940 bytes Optimized: yes PDF version: 1.3 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- FNGLNG+AdvOTea1a7398 Type 1C Custom yes yes yes 472 0 FNGLOG+AdvOT9bd21c25.I Type 1C Custom yes yes yes 480 0 FNGLOH+AdvOTcb88df00 Type 1C Custom yes yes yes 481 0 FNGMFG+Arial-BoldMT Type 1C WinAnsi yes yes no 482 0 FNGMJF+AdvOTf23bb480 Type 1C Custom yes yes yes 483 0 FNGMJG+AdvP4C4E74 Type 1C Custom yes yes yes 493 0 FNGNDE+AdvTTda6f6cb8.B Type 1C Custom yes yes yes 502 0 FNGNLG+AdvOT7d6df7ab.I Type 1C Custom yes yes yes 244 0 FNGNID+AdvOT125c650c Type 1C Custom yes yes yes 245 0 FNGNIE+AdvOT1ef757c0 Type 1C Custom yes yes yes 242 0 FNGNJE+AdvPi1 Type 1C WinAnsi yes yes no 257 0 FNGNKF+AdvOTb65e897d.B Type 1C Custom yes yes yes 258 0 FNHLIC+Helvetica Type 1C WinAnsi yes yes no 271 0 FNHLPA+Helvetica-Bold Type 1C WinAnsi yes yes no 268 0 FNHNDJ+AdvEls-ent4 Type 1C WinAnsi yes yes no 266 0 FNKGPK+Symbol Type 1C WinAnsi yes yes no 277 0 FNLBOI+AdvEls-ent5 Type 1C WinAnsi yes yes no 279 0 FNODPF+Advsymbol Type 1C Custom yes yes no 293 0 FNPCJM+AdvItcSymbol-M Type 1C WinAnsi yes yes no 297 0 FNPDFJ+AdvEls-ent3 Type 1C WinAnsi yes yes no 298 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-02-16 02:58:31 CET RepresentationInformation: pmc_27882925.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2018-02-06 08:44:17 CET Size: 3268940 Format: PDF Version: 1.3 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf Profile: Linearized PDF PDFMetadata: Objects: 525 FreeObjects: 1 IncrementalUpdates: 1 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: title_link Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Children: Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Children: Item: Title: ISGylation inhibits exosome secretion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: ISGylation decreases MVB numbers Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Figure™1ISGylation inhibits exosome secretion.(a) Western blot analysis of EVs purified by serial ultracentrifugation from cell culture supernatants from equal numbers of Jurkat T cells untreated (Cont) or treated with 1,000thinspUthinspml-1 IFN-I for 16t Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Figure™2ISGylation decreases MVB numbers.(a) Confocal microscopy analysis of the endosome markers HRS (red) and CD63 (green) in non-transfected HEK293 cells or HEK293 cells co-transfected with plasmids encoding the ISGylation machinery and the functional Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Children: Item: Title: ISGylation induces protein aggregation and degradation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Inhibition of MVB-lysosome fusion rescues exosome secretion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: ISG15 impairs exosome secretion by modifying TSG101 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Figure™3ISG15 conjugation induces protein aggregation and degradation by lysosomes.(a) Confocal co-localization analysis of ISG15-GFP (green) and the MVB marker HRS (red). Right graphs: fluorescence intensity profiles of ISG15-GFP (green) and HRS (red) in Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Figure™4Inhibition of lysosome function upon ISGylation induction rescues exosome secretion.(a) Western blot analysis of exosome secretion in untransfected HEK293 cells and HEK293 cells co-transfected with plasmids encoding the ISGylation machinery and IS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Children: Item: Title: Cell culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: Figure™5ISG15 impairs exosome secretion by modifying TSG101.(a) Western blot analysis of TSG101 immunoprecipitation in HEK293 cells non-transfected or HEK293 cells co-transfected with plasmids encoding the ISGylation machinery and functional (ISG15WT) or Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Children: Item: Title: Exosome purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Item: Title: Nanoparticle tracking analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@69b0fd6f Item: Title: ISGylation induction by overexpression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@757942a1 Item: Title: Exosome extraction from blood serum Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4a87761d Item: Title: Gene silencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@66d1af89 Item: Title: Western blotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@8646db9 Item: Title: Fluorescence confocal microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@37374a5e Item: Title: Cloning Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4671e53b Item: Title: Protein aggregation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@2db7a79b Item: Title: Protein degradation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6950e31 Item: Title: Immunoprecipitation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@b7dd107 Item: Title: Electron microscopy and MVB quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@42eca56e Item: Title: Figure™6Proposed model for the role of ISGylation in the regulation of exosome secretion.IFN-I induces ISG15 expression and conjugation to MVB proteins such as TSG101. ISGylation of MVB proteins promotes MVB fusion with lysosomes and degradation, thus inh Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@52f759d7 Children: Item: Title: Data availability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7cbd213e Item: Title: AndreolaG.Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesiclesJ. Exp. Med.195130313162002TheryC.Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomesNat. Immunol.3115611622002ChalminF.Membrane-asso Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@192d3247 Item: Title: We thank Dr K. Knobeloch, Dr A. García-Sastre and Dr M.A. Alonso for providing reagents, and Dr S. Bartlett for assistance with English editing. C.E. is thankful to electron microscopy facility (campus Casanova), CCiT-University of Barcelona. 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