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  • 96
    Thermo Fisher cd9
    Detection of LGTV RNA and proteins in exosomes isolated from N2a neuronal cell line. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 6; 72 h p.i.), N2a cells (1 x 10 7 ). Scale indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) N2a cell-derived exosomes. Number of exosomes analyzed were n = 32 (uninfected) and n = 131 (infected) groups. (D) Comparison of exosome numbers per image from uninfected (n = 9) and infected (n = 13) groups is shown. (E) DG-Exos showing presence of enhanced LGTV envelope [E]-protein loads in fractions 1–6. Fractions 3–5 showed enriched amounts of exosomal markers <t>CD9</t> and HSP70. E-protein detection in fraction 5 in 0.1 μm filtered samples processed for OptiPrep DG-isolation is shown. QRT-PCR analysis showing levels of total LGTV loads (F), copy numbers (G) and LGTV positive-sense strand or negative-sense strand (H) in exosomes isolated from N2a cells at different time points. N2a (1 x 10 5 ) cells were infected with 6 MOI of LGTV, and LGTV loads were analyzed at 72 h p.i. (I) Treatment of LGTV-infected (72 h p.i.) N2a cell-derived exosomes with RNase A is shown. The uninfected samples treated with RNase serve as control. LGTV transcript levels were normalized to mouse beta-actin. (J) Immunoblotting analysis showing detection of LGTV E glycoprotein and mammalian exosomal marker CD9 in exosome fractions and total lysates from whole cells prepared at 48 h p.i. from 2 x 10 6 uninfected (UI) or infected (I) N2a cells. Stain-free gels showing total protein profiles serve as the loading control. (K) Native-PAGE followed by immunoblotting analysis showing presence of LGTV E- and NS1 proteins from LGTV-infected (MOI 1; 72 h p.i.) or uninfected N2a cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated, held on ice. Coomassie stained gel image showing total protein profiles serve as a loading control. (L) ELISA performed on uninfected or LGTV-infected (MOI 6; 72 h p.i.) N2a cell-derived exosomes either untreated or treated with Triton-X-100 (0.1%). (M) Antibody-beads binding assay performed on LGTV infected (MOI 1, 72 h p.i.) N2a cell-derived exosomes (collected from N2a cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in LGTV-E protein loads between 4G2 or isotype and untreated samples.
    Cd9, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore anti cd9
    The level of total proteins in the sEVs is reduced in U bl 3 knockout mice. a The cell lysate (CL) and pellets from the conditioned medium of MDA-MB-231 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. b The presence of UBL3 and its mutants in sEVs. c Electron microscopic analyses of purified sEVs by negative staining. Scale bars, 100 nm. d Upper left panel, protein staining for the sEVs from the serum in WT and Ubl3 KO mice. Lower panels, purified serum sEVs were blotted with anti-tubulin and <t>CD9</t> antibodies with βME. Right panel, relative intensity of total proteins in serum sEVs. n = 9 pairs. e Total RNA levels in the serum sEVs. WT, n = 5; KO, n = 5. n.s., p > 0.05 by Mann–Whitney test. a,b, βME + condition: Flag, Flotillin-1, GP96, Actinin-4, Calreticulin, GAPDH, and Alix antibodies. βME- condition: CD63, and CD9 antibodies
    Anti Cd9, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd9  (Abcam)
    99
    Abcam cd9
    The level of total proteins in the sEVs is reduced in U bl 3 knockout mice. a The cell lysate (CL) and pellets from the conditioned medium of MDA-MB-231 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. b The presence of UBL3 and its mutants in sEVs. c Electron microscopic analyses of purified sEVs by negative staining. Scale bars, 100 nm. d Upper left panel, protein staining for the sEVs from the serum in WT and Ubl3 KO mice. Lower panels, purified serum sEVs were blotted with anti-tubulin and <t>CD9</t> antibodies with βME. Right panel, relative intensity of total proteins in serum sEVs. n = 9 pairs. e Total RNA levels in the serum sEVs. WT, n = 5; KO, n = 5. n.s., p > 0.05 by Mann–Whitney test. a,b, βME + condition: Flag, Flotillin-1, GP96, Actinin-4, Calreticulin, GAPDH, and Alix antibodies. βME- condition: CD63, and CD9 antibodies
    Cd9, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 785 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology cd9
    <t>CD9</t> is required for the gp130-mediated BMX-STAT3 signaling in GSCs. ( a , b ) Immunoblot analyses of CD9, gp130, p-BMX (p-Tyr40), BMX, p-STAT3 (p-Tyr705) and STAT3 in D456-GSCs ( a ) or T4121-GSCs ( b ) expressing shCD9 or shNT in combination with IL-6 treatment. Cells were treated with IL-6 (10 ng/ml) or control vehicle for 10 min and were collected for immunoblot analyses. CD9 disruption abolished the IL-6 induced activating phosphorylations of BMX (p-Tyr40) and STAT3 (p-Tyr705). ( c , d ) Immunoblot analyses of p-STAT3 (p-Tyr705), STAT3, Bcl-xL and c-Myc in D456-GSCs ( c ) or T4121-GSCs ( d ) expressing shCD9 or shNT. Silencing CD9 attenuated the activity of STAT3 (p-STAT3) and thus suppressed the expressions of STAT3 downstream targets Bcl-xL and c-Myc. All experiments were performed independently for three times
    Cd9, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson cd9
    MMP-9 expression and release are greatly enhanced in <t>CD9-HT1080</t> cells. ( A ) Fold changes in MMP and TIMP mRNA expression for Mock- and CD9-HT1080 cells were calculated from cycle threshold values using qRT-PCR. ( B, C ) Specific ELISA kits were used to measure the concentration (ng/ml) of pro- and active- MMP-1 and MMP-9 in the cleared supernatant of Mock- and CD9-HT1080 cells. ( D ) A representative gelatin zymogram of Mock- and CD9- HT1080 cell conditioned media. The absence of Coomassie staining at 92 kDa indicates the presence of pro-MMP-9 and at 72 kDa represents pro-MMP-2. ( E ) Quantification of the relative band intensity was calculated by densitometry analysis as described in the Materials and methods section *, p
    Cd9, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti cd9
    <t>Anti-CD9</t> mAb inhibits chemotaxis toward Ag and induces tyrosine phosphorylation of NTAL by different mechanism. A, IgE-sensitized BMMCs were pretreated with the indicated concentrations of anti-CD9 mAb 2H9 for 15 min and their chemotactic response toward
    Anti Cd9, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti cd9
    <t>CD9</t> is required for the gp130-mediated BMX-STAT3 signaling in GSCs. ( a , b ) Immunoblot analyses of CD9, gp130, p-BMX (p-Tyr40), BMX, p-STAT3 (p-Tyr705) and STAT3 in D456-GSCs ( a ) or T4121-GSCs ( b ) expressing shCD9 or shNT in combination with IL-6 treatment. Cells were treated with IL-6 (10 ng/ml) or control vehicle for 10 min and were collected for immunoblot analyses. CD9 disruption abolished the IL-6 induced activating phosphorylations of BMX (p-Tyr40) and STAT3 (p-Tyr705). ( c , d ) Immunoblot analyses of p-STAT3 (p-Tyr705), STAT3, Bcl-xL and c-Myc in D456-GSCs ( c ) or T4121-GSCs ( d ) expressing shCD9 or shNT. Silencing CD9 attenuated the activity of STAT3 (p-STAT3) and thus suppressed the expressions of STAT3 downstream targets Bcl-xL and c-Myc. All experiments were performed independently for three times
    Anti Cd9, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ACROBiosystems cd19
    The molecular characteristics of CAR-T and <t>CD19</t> + B cells. (A–B) Expression levels of the selected genes in CAR-T and CD19 + B cells before and after treatment were detected by RNA-seq. Left panel: the gene expression details of CAR-T or CD19 + B cells in pretreatment and post-treatment samples; right panel: the fold change in the expression of selected genes in post-treatment samples derived CAR-T or CD19 + B cells (relative to the pretreatment value). (C) The CD19 + B cells isolated from tumor tissues of relapsed patient exhibited a missense mutation in exon 3 (P. 163 R > L/C.488 G > T). (D) The autologous CAR (FMC63)-T cells cocultured with CD19 + B cells were derived from bone marrow before treatment and from tumor tissues after relapse at a ratio of 10:1. The CAR-T cells are potentiated to eradicate normal CD19 + cells, but not the mutated target cells. (E) The FMC63 and 21D4 CAR-T cells were incubated with target cells derived from tumor tissues. *p
    Cd19, supplied by ACROBiosystems, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc cd9
    Western blot analysis. Western blot was performed on cExo and ncExo samples, both from exosome-fractions (fractions 7–9) and from a non-exosome-fraction (fraction 14). Normal human keratinocyte lysate was used as control. (a) <t>CD9</t> was positive in all exosome-samples and in the lysate, but negative in the non-exosome-fraction. (b) Flotillin was positive in all exosome-fractions and lysate, but with varying signal strength, and no signal was detected in the non-exosome-fraction. (c) Annexin V was negative in all fractions, but positive in the lysate. (d) EpCAM was negative in all samples (expected at 40 kDa, dotted box). (e) HSP70, heat shock protein 70, was negative in all samples except for cell lysate control. (f) GRP94, an endoplasmic reticulum protein that commonly contaminates vesicle isolates, was also negative in all samples except for cell lysate control. d3, d4 and d5 refer to donors 3, 4 and 5.
    Cd9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher cd9 monoclonal antibody
    Flow cytometry and WB analysis targeting the exosome marker <t>CD9</t> on isolated vesicles. CD9 positive vesicles were detected by flow cytometry. Median fluorescence intensity (MFI) was reported as a signal to noise (S/N) ratio to isotype control in EVs isolated from E10 (A), BxPC3 (B) and H3 (C) cells (n = 3). The presence of CD9 was also analyzed by WB, which was detected in vesicles from E10 (D) and BxPC3 cells (E) (n = 3).
    Cd9 Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti cd9 antibody
    Flow cytometry and WB analysis targeting the exosome marker <t>CD9</t> on isolated vesicles. CD9 positive vesicles were detected by flow cytometry. Median fluorescence intensity (MFI) was reported as a signal to noise (S/N) ratio to isotype control in EVs isolated from E10 (A), BxPC3 (B) and H3 (C) cells (n = 3). The presence of CD9 was also analyzed by WB, which was detected in vesicles from E10 (D) and BxPC3 cells (E) (n = 3).
    Anti Cd9 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher anti cd9
    Exosomes from patients with MS can selectively decrease the frequency of Treg cells a Representative size distribution of purified exosomes. Using a NanoSight LM10 nanoparticle analysis system, the size was analysed three times for each sample. Red error bars indicate the standard error of the mean. b Western blot analysis for <t>CD9,</t> CD63 and Cytochrome c proteins. The PBMC and exosome samples were collected from HC. Each lane was loaded with 3 μg of protein for blotting. c Dot plot and histogram of flow cytometry data. T cells were prepared from the peripheral blood of a healthy volunteer. They were cultured with PBS as a control or with exosomes derived from HC (HC-exosome) or patients with MS (MS-exosome) under stimulation with anti-CD3 and anti-CD28 mAbs for 48 h. IFN-γ − IL-17A − CD4 + T cells were defined as shown in the left panel, and then the expression of Foxp3 was evaluated as shown in the right panel. d The frequencies of inflammatory and regulatory T cells among CD4 + T cells after the culture described above. Among Foxp3 + CD4 + T cells, IFN-γ − IL-17A − Foxp3 + CD4 + , whereas Foxp3 + CD4 + . Data are representative of two independent experiments. A one-way ANOVA with Bonferroni’s comparison test was used for statistical analysis. Error bars represent the mean ± s.d. * p
    Anti Cd9, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    System Biosciences Inc anti cd9
    Exosomes containing viral RNA induce NKG2D ligand expression in macrophages . (A) HepG2 cells were seeded onto a 24-well plate and cultured for 24 h. EVs were isolated from 0.5 ml of cell culture medium using a polyethylene glycol method with exosome isolation kit (see Isolation of Exosomes in Experimental Procedures ) and suspended with 150 μl of 1× SDS sample buffer. The 150 μl of whole cell extract (WCE) were prepared from cultured cells. The 10 μl of EVs and 10 μl of WCE were subjected to SDS-PAGE. <t>CD9</t> and β-actin proteins were detected by western blotting with anti-CD9 antibody and anti-β-actin antibody. (B) Extracellular vesicles (EVs) released from HepG2 (left) or HuH-7 (right) transfected with pHBV were collected, and the total RNA was extracted. The RNA levels of HBV RNA and GAPDH mRNA were determined by RT-qPCR and normalized against that of U6 RNA. Data are presented as mean ± SD. (C) Exosomes were isolated from EVs released from HepG2 cells using anti-CD81 antibody beads. WCE, EVs, and 10× concentrated exosomes were subjected to SDS-PAGE, and the proteins were detected by western blotting. (D) The CD81 + exosomes were isolated from EVs with anti-CD81 microbeads. The RNA levels were determined as described in (B) . Data are presented as mean ± SD ( n = 3). (E) Hepatic F4/80 + cells and hepatic NK cells were co-cultured with normal HepG2 cells or HepG2-T23 cells (HepG2-HBV), which stably express HBV, for 1 day. IFN-γ levels in the culture supernatants were determined using ELISA ( n > 3). (F) EVs released from HuH-7 or HepG2 with or without HBV were added to PMA-treated THP-1 cells (THP-1 macrophages) for 24 h. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR and normalized to GAPDH ( n = 3). (G) HepG2 cells in six-well plates were transfected with mock or pHBV and were cultured for 24 h. EVs were isolated from 5 ml of culture medium and were suspended with 100 μl of PBS. The 5 μl of EVs were mixed with 5 μl of 2× SDS sample buffer and were subjected to SDS-PAGE. The proteins were detected by western blotting with anti-CD63 antibody. (H) EVs released from HepG2 with or without HBV were added to mouse hepatic F4/80 + cells for 24 h. The expression of mRNA in hepatic F4/80 + cells was determined by RT-qPCR and normalized to GAPDH ( n = 3). (I) HepG2-NTCP cells were infected with HBV for 9 days and were subsequently co-cultured with THP-1 (transwell co-culture) for 3 days. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR.
    Anti Cd9, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 93/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    System Biosciences Inc cd9
    Membrane CD163 is associated with exosomes. ( A ) Beads-based FACS analysis of exosome-FITC and <t>CD9-PE</t> staining on αCD163 (dark grey) or Isotype IgG immuno-precipitated EV’s isolated from human plasma. ( B ) Negative-stain EM of exosomes purified by αCD163 or αCD9 immunoprecipitation of EV’s isolated from human plasma.
    Cd9, supplied by System Biosciences Inc, used in various techniques. Bioz Stars score: 93/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti cd9
    Membrane CD163 is associated with exosomes. ( A ) Beads-based FACS analysis of exosome-FITC and <t>CD9-PE</t> staining on αCD163 (dark grey) or Isotype IgG immuno-precipitated EV’s isolated from human plasma. ( B ) Negative-stain EM of exosomes purified by αCD163 or αCD9 immunoprecipitation of EV’s isolated from human plasma.
    Anti Cd9, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam antibodies against cd9
    Isolation of exosomes secreted from gastric cancer cells SGC-7901 and BGC-823 treated by irradiation. A : Electron microscopic images of exosomes released from SGC-7901 and BGC-823 cell lines. Exosomes were isolated 24 h after irradiation at either 0 Gy or 6 Gy. Bars = 0.2μm. B : Western blot analysis of <t>CD9</t> protein in cell lysates prepared from exosomes of SGC-7901 or BGC-823 cell line. Whole cell extract from SGC-7901 or BGC823 cells was used as negative control (NC) and GAPDH was detected as loading control.
    Antibodies Against Cd9, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson mouse anti human cd9
    Direct identification of exosomes in CCS. (a) Detection of <t>CD9</t> and CD41b in CCS and CCS filtrate by western blot. Cell lysates were used as a positive control. The proteins were concentrated by trichloroacetic acid (TCA) from CCS/CCS filtrate. (b) SPRi response signals of anti-CD9/CD41b and anti-mouse IgG to cell culture medium (DMEM), CCS, and CCS filtrate. The results are representative of three independent experiments. * p
    Mouse Anti Human Cd9, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Immunostep anti cd9
    Optimization of tetraspanin antibody combination. 4∙10 9 particles of PC3-derived EVs were captured onto either <t>anti-CD9</t> (Clone VJ1/20) or anti-CD63 (Clone TEA3/18) coated beads followed by detection with PE-conjugated antibodies directed against CD9, CD81 or CD63. The sensitivity of each antibody combination is compared using the Stain Index (SI) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$SI=\frac{MFI\,positive\mbox{--}MFI\,background}{2\sigma \,background}$$\end{document} S I = M F I p o s i t i v e – M F I b a c k g r o u n d 2 σ b a c k g r o u n d , where σ is the standard deviation and MFI Mean Fluorescence Intensity; and the Relative fluorescence Index (RFI), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RFI=\frac{MFI\,positive}{MFI\,background}$$\end{document} R F I = M F I p o s i t i v e M F I b a c k g r o u n d , indicated on the upper right corner of each panel. A representative experiment out of 3 is shown.
    Anti Cd9, supplied by Immunostep, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Detection of LGTV RNA and proteins in exosomes isolated from N2a neuronal cell line. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 6; 72 h p.i.), N2a cells (1 x 10 7 ). Scale indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) N2a cell-derived exosomes. Number of exosomes analyzed were n = 32 (uninfected) and n = 131 (infected) groups. (D) Comparison of exosome numbers per image from uninfected (n = 9) and infected (n = 13) groups is shown. (E) DG-Exos showing presence of enhanced LGTV envelope [E]-protein loads in fractions 1–6. Fractions 3–5 showed enriched amounts of exosomal markers CD9 and HSP70. E-protein detection in fraction 5 in 0.1 μm filtered samples processed for OptiPrep DG-isolation is shown. QRT-PCR analysis showing levels of total LGTV loads (F), copy numbers (G) and LGTV positive-sense strand or negative-sense strand (H) in exosomes isolated from N2a cells at different time points. N2a (1 x 10 5 ) cells were infected with 6 MOI of LGTV, and LGTV loads were analyzed at 72 h p.i. (I) Treatment of LGTV-infected (72 h p.i.) N2a cell-derived exosomes with RNase A is shown. The uninfected samples treated with RNase serve as control. LGTV transcript levels were normalized to mouse beta-actin. (J) Immunoblotting analysis showing detection of LGTV E glycoprotein and mammalian exosomal marker CD9 in exosome fractions and total lysates from whole cells prepared at 48 h p.i. from 2 x 10 6 uninfected (UI) or infected (I) N2a cells. Stain-free gels showing total protein profiles serve as the loading control. (K) Native-PAGE followed by immunoblotting analysis showing presence of LGTV E- and NS1 proteins from LGTV-infected (MOI 1; 72 h p.i.) or uninfected N2a cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated, held on ice. Coomassie stained gel image showing total protein profiles serve as a loading control. (L) ELISA performed on uninfected or LGTV-infected (MOI 6; 72 h p.i.) N2a cell-derived exosomes either untreated or treated with Triton-X-100 (0.1%). (M) Antibody-beads binding assay performed on LGTV infected (MOI 1, 72 h p.i.) N2a cell-derived exosomes (collected from N2a cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in LGTV-E protein loads between 4G2 or isotype and untreated samples.

    Journal: PLoS Pathogens

    Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

    doi: 10.1371/journal.ppat.1006764

    Figure Lengend Snippet: Detection of LGTV RNA and proteins in exosomes isolated from N2a neuronal cell line. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 6; 72 h p.i.), N2a cells (1 x 10 7 ). Scale indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) N2a cell-derived exosomes. Number of exosomes analyzed were n = 32 (uninfected) and n = 131 (infected) groups. (D) Comparison of exosome numbers per image from uninfected (n = 9) and infected (n = 13) groups is shown. (E) DG-Exos showing presence of enhanced LGTV envelope [E]-protein loads in fractions 1–6. Fractions 3–5 showed enriched amounts of exosomal markers CD9 and HSP70. E-protein detection in fraction 5 in 0.1 μm filtered samples processed for OptiPrep DG-isolation is shown. QRT-PCR analysis showing levels of total LGTV loads (F), copy numbers (G) and LGTV positive-sense strand or negative-sense strand (H) in exosomes isolated from N2a cells at different time points. N2a (1 x 10 5 ) cells were infected with 6 MOI of LGTV, and LGTV loads were analyzed at 72 h p.i. (I) Treatment of LGTV-infected (72 h p.i.) N2a cell-derived exosomes with RNase A is shown. The uninfected samples treated with RNase serve as control. LGTV transcript levels were normalized to mouse beta-actin. (J) Immunoblotting analysis showing detection of LGTV E glycoprotein and mammalian exosomal marker CD9 in exosome fractions and total lysates from whole cells prepared at 48 h p.i. from 2 x 10 6 uninfected (UI) or infected (I) N2a cells. Stain-free gels showing total protein profiles serve as the loading control. (K) Native-PAGE followed by immunoblotting analysis showing presence of LGTV E- and NS1 proteins from LGTV-infected (MOI 1; 72 h p.i.) or uninfected N2a cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated, held on ice. Coomassie stained gel image showing total protein profiles serve as a loading control. (L) ELISA performed on uninfected or LGTV-infected (MOI 6; 72 h p.i.) N2a cell-derived exosomes either untreated or treated with Triton-X-100 (0.1%). (M) Antibody-beads binding assay performed on LGTV infected (MOI 1, 72 h p.i.) N2a cell-derived exosomes (collected from N2a cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in LGTV-E protein loads between 4G2 or isotype and untreated samples.

    Article Snippet: For DG-Exos samples, equal volume (20 μl) of each fraction from 1–6 or 20 μg of total protein lysates from uninfected and infected cells or 10 μl of each fraction from virus stock samples were loaded onto 12% SDS-PAGE, followed by immunoblotting and labeling with highly cross-reactive 4G2 monoclonal antibody to detect LGTV E-protein or exosomal specific markers such as HSP70 (rabbit polyclonal; Cell Signaling Technologies, Inc) or CD9 (mouse monoclonal; Invitrogen/ThermoScientific) and respective secondary antibodies (Santa Cruz Biotechnologies, Inc).

    Techniques: Isolation, Infection, Derivative Assay, Quantitative RT-PCR, Marker, Staining, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Binding Assay

    Detection of LGTV RNA and proteins in exosomes isolated from primary cultures of mouse cortical neurons. LGTV (4 MOI) was used to infect 1 x 10 5 murine cortical neuronal cells. UI indicates uninfected and I indicates LGTV-infected. (A) QRT-PCR analysis showing LGTV infection kinetics in primary cultures of mouse cortical neurons at different time points (24, 48, 72, 96 h p.i.). Total LGTV loads in exosomes isolated from cortical neurons (B), copy numbers (C) and levels of LGTV positive-sense strand or negative-sense strand (D) in exosomes isolated from cortical neuronal cells at different time points (24, 48 and 72 h p.i.). (E) Immunoblotting analysis showing detection of LGTV E-protein and mammalian exosomal marker CD9 in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) cortical neuronal cells at 48 h p.i. Stain-free gel showing total protein profile serves as the loading control. (F) Immunoblotting analysis showing NS1 levels in total neuronal lysates and exosomes derived from LGTV-infected (MOI 4; 72 h p.i.) cortical neurons. Uninfected (UI) cells and exosomes derived from these cells serve as controls in addition to total protein profiles. For immunoblotting assays, 2 x 10 7 cortical neuronal cells were infected with 4 MOI of LGTV. (G) Quantitative assessment of number of plaques from exosomal and supernatant fractions is shown. (H) QRT-PCR analysis of viral loads in 1x 10 5 naïve cortical neuronal cells at 24 h p.i., infected through treatment with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 24, 48 and 72 h p.i., LGTV-infected neurons show presence of LGTV. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tailed t test is shown. Representative data is shown from at least three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

    doi: 10.1371/journal.ppat.1006764

    Figure Lengend Snippet: Detection of LGTV RNA and proteins in exosomes isolated from primary cultures of mouse cortical neurons. LGTV (4 MOI) was used to infect 1 x 10 5 murine cortical neuronal cells. UI indicates uninfected and I indicates LGTV-infected. (A) QRT-PCR analysis showing LGTV infection kinetics in primary cultures of mouse cortical neurons at different time points (24, 48, 72, 96 h p.i.). Total LGTV loads in exosomes isolated from cortical neurons (B), copy numbers (C) and levels of LGTV positive-sense strand or negative-sense strand (D) in exosomes isolated from cortical neuronal cells at different time points (24, 48 and 72 h p.i.). (E) Immunoblotting analysis showing detection of LGTV E-protein and mammalian exosomal marker CD9 in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) cortical neuronal cells at 48 h p.i. Stain-free gel showing total protein profile serves as the loading control. (F) Immunoblotting analysis showing NS1 levels in total neuronal lysates and exosomes derived from LGTV-infected (MOI 4; 72 h p.i.) cortical neurons. Uninfected (UI) cells and exosomes derived from these cells serve as controls in addition to total protein profiles. For immunoblotting assays, 2 x 10 7 cortical neuronal cells were infected with 4 MOI of LGTV. (G) Quantitative assessment of number of plaques from exosomal and supernatant fractions is shown. (H) QRT-PCR analysis of viral loads in 1x 10 5 naïve cortical neuronal cells at 24 h p.i., infected through treatment with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 24, 48 and 72 h p.i., LGTV-infected neurons show presence of LGTV. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tailed t test is shown. Representative data is shown from at least three independent experiments.

    Article Snippet: For DG-Exos samples, equal volume (20 μl) of each fraction from 1–6 or 20 μg of total protein lysates from uninfected and infected cells or 10 μl of each fraction from virus stock samples were loaded onto 12% SDS-PAGE, followed by immunoblotting and labeling with highly cross-reactive 4G2 monoclonal antibody to detect LGTV E-protein or exosomal specific markers such as HSP70 (rabbit polyclonal; Cell Signaling Technologies, Inc) or CD9 (mouse monoclonal; Invitrogen/ThermoScientific) and respective secondary antibodies (Santa Cruz Biotechnologies, Inc).

    Techniques: Isolation, Infection, Quantitative RT-PCR, Marker, Staining, Derivative Assay, Two Tailed Test

    The level of total proteins in the sEVs is reduced in U bl 3 knockout mice. a The cell lysate (CL) and pellets from the conditioned medium of MDA-MB-231 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. b The presence of UBL3 and its mutants in sEVs. c Electron microscopic analyses of purified sEVs by negative staining. Scale bars, 100 nm. d Upper left panel, protein staining for the sEVs from the serum in WT and Ubl3 KO mice. Lower panels, purified serum sEVs were blotted with anti-tubulin and CD9 antibodies with βME. Right panel, relative intensity of total proteins in serum sEVs. n = 9 pairs. e Total RNA levels in the serum sEVs. WT, n = 5; KO, n = 5. n.s., p > 0.05 by Mann–Whitney test. a,b, βME + condition: Flag, Flotillin-1, GP96, Actinin-4, Calreticulin, GAPDH, and Alix antibodies. βME- condition: CD63, and CD9 antibodies

    Journal: Nature Communications

    Article Title: UBL3 modification influences protein sorting to small extracellular vesicles

    doi: 10.1038/s41467-018-06197-y

    Figure Lengend Snippet: The level of total proteins in the sEVs is reduced in U bl 3 knockout mice. a The cell lysate (CL) and pellets from the conditioned medium of MDA-MB-231 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. b The presence of UBL3 and its mutants in sEVs. c Electron microscopic analyses of purified sEVs by negative staining. Scale bars, 100 nm. d Upper left panel, protein staining for the sEVs from the serum in WT and Ubl3 KO mice. Lower panels, purified serum sEVs were blotted with anti-tubulin and CD9 antibodies with βME. Right panel, relative intensity of total proteins in serum sEVs. n = 9 pairs. e Total RNA levels in the serum sEVs. WT, n = 5; KO, n = 5. n.s., p > 0.05 by Mann–Whitney test. a,b, βME + condition: Flag, Flotillin-1, GP96, Actinin-4, Calreticulin, GAPDH, and Alix antibodies. βME- condition: CD63, and CD9 antibodies

    Article Snippet: Briefly, before performing the immuno-isolation, anti-CD9 (Millipore, MM2/57, 1:10), anti-CD63 (BD, H5C6, 1:50) and control mouse polyclonal IgG (Millipore, 12-371, 1:100) incubated with 100 μL of protein A magnetic beads (Thermo, 88845) for 16 h at 4 °C with rotation.

    Techniques: Knock-Out, Mouse Assay, Multiple Displacement Amplification, Transfection, Purification, Negative Staining, Staining, MANN-WHITNEY

    AZIN2 over-expressing T84 colon cancer cells release CD63 positive exosomes. Immunoblotting of cell lysates (15μg) and of exosomes (EV) (2 μg) isolated by ultracentrifugation from culture supernatant of AZIN2 cDNA over-expressing and control T84 colon cancer cells transfected with the empty vector using antibodies to HSP90a/b, CD63 and CD9.

    Journal: PLoS ONE

    Article Title: Ornithine decarboxylase antizyme inhibitor 2 (AZIN2) is a signature of secretory phenotype and independent predictor of adverse prognosis in colorectal cancer

    doi: 10.1371/journal.pone.0211564

    Figure Lengend Snippet: AZIN2 over-expressing T84 colon cancer cells release CD63 positive exosomes. Immunoblotting of cell lysates (15μg) and of exosomes (EV) (2 μg) isolated by ultracentrifugation from culture supernatant of AZIN2 cDNA over-expressing and control T84 colon cancer cells transfected with the empty vector using antibodies to HSP90a/b, CD63 and CD9.

    Article Snippet: Other primary antibodies used were mouse monoclonal anti-human GAPDH (G8795), mouse monoclonal anti-human CD63 (SAB4700215) and rabbit polyclonal anti-human CD9 (SAB4503606), all from Sigma-Aldrich; HSP90α/β (sc-13119) was from Santa Cruz Biotechnology.

    Techniques: Expressing, Isolation, Transfection, Plasmid Preparation

    CD9 is required for the gp130-mediated BMX-STAT3 signaling in GSCs. ( a , b ) Immunoblot analyses of CD9, gp130, p-BMX (p-Tyr40), BMX, p-STAT3 (p-Tyr705) and STAT3 in D456-GSCs ( a ) or T4121-GSCs ( b ) expressing shCD9 or shNT in combination with IL-6 treatment. Cells were treated with IL-6 (10 ng/ml) or control vehicle for 10 min and were collected for immunoblot analyses. CD9 disruption abolished the IL-6 induced activating phosphorylations of BMX (p-Tyr40) and STAT3 (p-Tyr705). ( c , d ) Immunoblot analyses of p-STAT3 (p-Tyr705), STAT3, Bcl-xL and c-Myc in D456-GSCs ( c ) or T4121-GSCs ( d ) expressing shCD9 or shNT. Silencing CD9 attenuated the activity of STAT3 (p-STAT3) and thus suppressed the expressions of STAT3 downstream targets Bcl-xL and c-Myc. All experiments were performed independently for three times

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: CD9 is required for the gp130-mediated BMX-STAT3 signaling in GSCs. ( a , b ) Immunoblot analyses of CD9, gp130, p-BMX (p-Tyr40), BMX, p-STAT3 (p-Tyr705) and STAT3 in D456-GSCs ( a ) or T4121-GSCs ( b ) expressing shCD9 or shNT in combination with IL-6 treatment. Cells were treated with IL-6 (10 ng/ml) or control vehicle for 10 min and were collected for immunoblot analyses. CD9 disruption abolished the IL-6 induced activating phosphorylations of BMX (p-Tyr40) and STAT3 (p-Tyr705). ( c , d ) Immunoblot analyses of p-STAT3 (p-Tyr705), STAT3, Bcl-xL and c-Myc in D456-GSCs ( c ) or T4121-GSCs ( d ) expressing shCD9 or shNT. Silencing CD9 attenuated the activity of STAT3 (p-STAT3) and thus suppressed the expressions of STAT3 downstream targets Bcl-xL and c-Myc. All experiments were performed independently for three times

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Activity Assay

    Expression of a STAT3-C compromises GSC maintenance impaired by CD9 disruption. ( a ) Immunoblot analyses of Flag-tagged STAT3-C, STAT3 and CD9 in D456 GSCs transduced with STAT3-C or vector and shCD9 or shNT control. ( b – d ) The representative images ( b ) and quantification of sphere numbers ( c ) and sphere diameters ( d ) of D456 GSCs expressing shCD9 and/or STAT3-C. Scale bar=100 μ m. STAT3-C compromised the inhibitory effect of CD9 disruption on GSC tumorsphere formation. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Expression of a STAT3-C compromises GSC maintenance impaired by CD9 disruption. ( a ) Immunoblot analyses of Flag-tagged STAT3-C, STAT3 and CD9 in D456 GSCs transduced with STAT3-C or vector and shCD9 or shNT control. ( b – d ) The representative images ( b ) and quantification of sphere numbers ( c ) and sphere diameters ( d ) of D456 GSCs expressing shCD9 and/or STAT3-C. Scale bar=100 μ m. STAT3-C compromised the inhibitory effect of CD9 disruption on GSC tumorsphere formation. ** P

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Transduction, Plasmid Preparation

    CD9 interacts with gp130 to mediate its function in GSCs. ( a ) MS analysis identified gp130 as the key interacting protein of CD9. CD9 was immunoprecipitated from T4121 GSCs expressing CD9-Flag using anti-Flag-conjugated beads. Gp130 was identified through MS analysis by peptides covering the gp130 protein sequence. A representative detected peptide (TNHFTIPK) of gp130 was shown. ( b ) Co-immunoprecipitation of gp130 with CD9 using anti-Flag-conjugated beads in D456 and T4121 GSCs transduced with CD9-Flag or vector control. ( c ) Representative immunofluorescent images of CD9 (in green) and gp130 (in red) in D456 and T4121 GSCs. Co-localization of CD9 and gp130 was detected in GSCs. Scale bar represents 10 μ m. ( d ) Quantification of co-localization rate of CD9 and gp130 in c . The co-localization rate of CD9 with gp130 was determined with five randomly selected images using Leica LAS AF Lite software. ( e ) Immunoblot analysis of gp130 in GSCs expressing shgp130 or shNT. Disrupting gp130 by shRNA effectively reduced gp130 expression. ( f ) Cell proliferation assay of GSCs expressing shgp130 or shNT. Gp130 disruption significantly suppressed GSC proliferation. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: CD9 interacts with gp130 to mediate its function in GSCs. ( a ) MS analysis identified gp130 as the key interacting protein of CD9. CD9 was immunoprecipitated from T4121 GSCs expressing CD9-Flag using anti-Flag-conjugated beads. Gp130 was identified through MS analysis by peptides covering the gp130 protein sequence. A representative detected peptide (TNHFTIPK) of gp130 was shown. ( b ) Co-immunoprecipitation of gp130 with CD9 using anti-Flag-conjugated beads in D456 and T4121 GSCs transduced with CD9-Flag or vector control. ( c ) Representative immunofluorescent images of CD9 (in green) and gp130 (in red) in D456 and T4121 GSCs. Co-localization of CD9 and gp130 was detected in GSCs. Scale bar represents 10 μ m. ( d ) Quantification of co-localization rate of CD9 and gp130 in c . The co-localization rate of CD9 with gp130 was determined with five randomly selected images using Leica LAS AF Lite software. ( e ) Immunoblot analysis of gp130 in GSCs expressing shgp130 or shNT. Disrupting gp130 by shRNA effectively reduced gp130 expression. ( f ) Cell proliferation assay of GSCs expressing shgp130 or shNT. Gp130 disruption significantly suppressed GSC proliferation. ** P

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Mass Spectrometry, Immunoprecipitation, Expressing, Sequencing, Transduction, Plasmid Preparation, Software, shRNA, Proliferation Assay

    Expression of the STAT-C restores GSC-driven tumor propagation impaired by CD9 disruption. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 and/or STAT3-C at day 17 and day 24 after tumor implantation. Data are shown as mean±S.E.M. ( b ). ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Expression of the STAT-C restores GSC-driven tumor propagation impaired by CD9 disruption. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 and/or STAT3-C at day 17 and day 24 after tumor implantation. Data are shown as mean±S.E.M. ( b ). ** P

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Derivative Assay, Tumor Implantation

    A schematic diagram shows that CD9 binds to gp130 and stabilizes gp130 by blocking its ubiquitin-dependent lysosomal degradation to promote the BMX-mediated STAT3 activation in GSCs. The binding of CD9 to gp130 prevents gp130 from ubiquitination and lysosomal degradation, thus facilitates the signal transduction of the IL-6-gp130-BMX-STAT3 signaling pathway in GSCs to maintain GSC self-renewal and tumorigenic potential

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: A schematic diagram shows that CD9 binds to gp130 and stabilizes gp130 by blocking its ubiquitin-dependent lysosomal degradation to promote the BMX-mediated STAT3 activation in GSCs. The binding of CD9 to gp130 prevents gp130 from ubiquitination and lysosomal degradation, thus facilitates the signal transduction of the IL-6-gp130-BMX-STAT3 signaling pathway in GSCs to maintain GSC self-renewal and tumorigenic potential

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Blocking Assay, Activation Assay, Binding Assay, Transduction

    CD9 stabilizes gp130 by preventing the ubiquitin-dependent lysosomal degradation. ( a , b ) qRT-PCR analysis of CD9 ( a ) and IL6ST (gene encoding for gp130) ( b ) in D456 and T4121 GSCs expressing shCD9 or shNT. Silencing CD9 did not affect mRNA level of gp130 in GSCs. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: CD9 stabilizes gp130 by preventing the ubiquitin-dependent lysosomal degradation. ( a , b ) qRT-PCR analysis of CD9 ( a ) and IL6ST (gene encoding for gp130) ( b ) in D456 and T4121 GSCs expressing shCD9 or shNT. Silencing CD9 did not affect mRNA level of gp130 in GSCs. ** P

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Quantitative RT-PCR, Expressing

    Tetraspanin CD9 is preferentially expressed in GSCs and is essential for the GSC maintenance. ( a ) The expression heatmap of tetraspanins in GSC lines ( n =12) relative to CGCs ( n =32) from the GEO profiles (GEO: GDS3885). Four candidates, including CD9 , TSPAN7 , TSPAN11 and TSPAN33 were significantly upregulated in GSCs relative to CGCs. Data were visualized using Cluster/Java Treeview. ( b ) Immunoblot analysis showing the preferential expressions of CD9 and the GSC marker SOX2 in GSCs ( n =6) relative to the matched NSTCs ( n =6) isolated from human GBMs. ( c ) Immunofluorescent staining of CD9 (in green) and the GSC marker SOX2 (in red, upper panel), OLIG2 (in red, middle panel) or CD133 (in red, lower panel) in GSC tumorspheres. Scale bar represents 25 μ m. ( d ) Immunoblot analyses of CD9, the GSC marker SOX2 and the neuronal differentiation marker MAP2 during the serum-induced differentiation of GSCs. The levels of CD9 and the GSC marker SOX2 decreased, while the expression of the differentiation marker MAP2 concomitantly increased over a 7-day period. ( e ) In vitro limiting dilution analyses of the secondary tumorsphere formations of GSCs expressing shCD9 (shCD9-1 and -2) or non-targeting shRNA (shNT, control). Disrupting CD9 expression attenuated the self-renewal capacity of GSCs. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Tetraspanin CD9 is preferentially expressed in GSCs and is essential for the GSC maintenance. ( a ) The expression heatmap of tetraspanins in GSC lines ( n =12) relative to CGCs ( n =32) from the GEO profiles (GEO: GDS3885). Four candidates, including CD9 , TSPAN7 , TSPAN11 and TSPAN33 were significantly upregulated in GSCs relative to CGCs. Data were visualized using Cluster/Java Treeview. ( b ) Immunoblot analysis showing the preferential expressions of CD9 and the GSC marker SOX2 in GSCs ( n =6) relative to the matched NSTCs ( n =6) isolated from human GBMs. ( c ) Immunofluorescent staining of CD9 (in green) and the GSC marker SOX2 (in red, upper panel), OLIG2 (in red, middle panel) or CD133 (in red, lower panel) in GSC tumorspheres. Scale bar represents 25 μ m. ( d ) Immunoblot analyses of CD9, the GSC marker SOX2 and the neuronal differentiation marker MAP2 during the serum-induced differentiation of GSCs. The levels of CD9 and the GSC marker SOX2 decreased, while the expression of the differentiation marker MAP2 concomitantly increased over a 7-day period. ( e ) In vitro limiting dilution analyses of the secondary tumorsphere formations of GSCs expressing shCD9 (shCD9-1 and -2) or non-targeting shRNA (shNT, control). Disrupting CD9 expression attenuated the self-renewal capacity of GSCs. ** P

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Marker, Isolation, Staining, In Vitro, shRNA

    Disrupting CD9 inhibits GSC-driven tumor growth and STAT3 activation in vivo. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 or shNT at day 14 and day 28 after tumor implantation. Silencing CD9 markedly suppressed GSC-driven tumor growth. * P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Disrupting CD9 inhibits GSC-driven tumor growth and STAT3 activation in vivo. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 or shNT at day 14 and day 28 after tumor implantation. Silencing CD9 markedly suppressed GSC-driven tumor growth. * P

    Article Snippet: Total cell lysates (input) were also immunoblotted with antibodies against gp130 (sc-655, 1:1000, Santa Cruz), CD9 (sc-13118, 1:1000, Santa Cruz) and tubulin (T9026, 1:10 000, Sigma).

    Techniques: Activation Assay, In Vivo, Derivative Assay, Expressing, Tumor Implantation

    MMP-9 expression and release are greatly enhanced in CD9-HT1080 cells. ( A ) Fold changes in MMP and TIMP mRNA expression for Mock- and CD9-HT1080 cells were calculated from cycle threshold values using qRT-PCR. ( B, C ) Specific ELISA kits were used to measure the concentration (ng/ml) of pro- and active- MMP-1 and MMP-9 in the cleared supernatant of Mock- and CD9-HT1080 cells. ( D ) A representative gelatin zymogram of Mock- and CD9- HT1080 cell conditioned media. The absence of Coomassie staining at 92 kDa indicates the presence of pro-MMP-9 and at 72 kDa represents pro-MMP-2. ( E ) Quantification of the relative band intensity was calculated by densitometry analysis as described in the Materials and methods section *, p

    Journal: PLoS ONE

    Article Title: Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

    doi: 10.1371/journal.pone.0067766

    Figure Lengend Snippet: MMP-9 expression and release are greatly enhanced in CD9-HT1080 cells. ( A ) Fold changes in MMP and TIMP mRNA expression for Mock- and CD9-HT1080 cells were calculated from cycle threshold values using qRT-PCR. ( B, C ) Specific ELISA kits were used to measure the concentration (ng/ml) of pro- and active- MMP-1 and MMP-9 in the cleared supernatant of Mock- and CD9-HT1080 cells. ( D ) A representative gelatin zymogram of Mock- and CD9- HT1080 cell conditioned media. The absence of Coomassie staining at 92 kDa indicates the presence of pro-MMP-9 and at 72 kDa represents pro-MMP-2. ( E ) Quantification of the relative band intensity was calculated by densitometry analysis as described in the Materials and methods section *, p

    Article Snippet: A rabbit polyclonal antibody specific for the first extracellular loop of CD9 (Rap2) was also generated in our laboratory and previously reported .Anti-CD63 and anti-CD151 antibodies were purchased from BD Pharmingen (San Diego, CA).

    Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining

    An increase in MMP-9 expression and release and subsequent cell invasion requires the second extracellular loop (EC2) of CD9. ( A ) MMP-9 mRNA expression was measured in Mock-, CD9-, and Δ6-HT1080 cells using qRT-PCR ( B,C ) Release of pro-MMP-9 and pro-MMP-2 was measured by gelatin zymography and quantified using Image J. ( D,E ) A representative picture of cell invasion through matrigel coated inserts and relative quantification of cell invasion *, p

    Journal: PLoS ONE

    Article Title: Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

    doi: 10.1371/journal.pone.0067766

    Figure Lengend Snippet: An increase in MMP-9 expression and release and subsequent cell invasion requires the second extracellular loop (EC2) of CD9. ( A ) MMP-9 mRNA expression was measured in Mock-, CD9-, and Δ6-HT1080 cells using qRT-PCR ( B,C ) Release of pro-MMP-9 and pro-MMP-2 was measured by gelatin zymography and quantified using Image J. ( D,E ) A representative picture of cell invasion through matrigel coated inserts and relative quantification of cell invasion *, p

    Article Snippet: A rabbit polyclonal antibody specific for the first extracellular loop of CD9 (Rap2) was also generated in our laboratory and previously reported .Anti-CD63 and anti-CD151 antibodies were purchased from BD Pharmingen (San Diego, CA).

    Techniques: Expressing, Quantitative RT-PCR, Zymography

    Silencing MMP-9 in CD9-HT1080 cells is sufficient to suppress the invasive phenotype. CD9-HT1080 cells were transiently transfected with short-interfering RNA directed to MMP-9 (CD9+ MMP-9 siRNA). Likewise, Mock- and CD9- HT1080 cells were transfected with a scrambled siRNA as a negative control (Mock+Ctr siRNA or CD9+ Ctr siRNA). ( A ) qRT-PCR analysis was used to measure changes in MMP-9 mRNA levels among Ctr and MMP-9 siRNA transfected cells. ( B ) Changes in release of MMP-9 (ng/ml) in to the cleared culture supernatant were examined using a MMP-9 specific ELISA kit. ( C ) Pro-MMP-9 levels in the supernatants of transiently transfected cells was measured using gelatin zymography and ( D ) quantified by densitometry. ( E ) A representative image of cells invading though matrigel after transfection with siRNA. ( F ) Percent cell invasion through matrigel-coated inserts after 20 hours was quantified and the results were normalized to Mock+Ctr siRNA treated cells *, p

    Journal: PLoS ONE

    Article Title: Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

    doi: 10.1371/journal.pone.0067766

    Figure Lengend Snippet: Silencing MMP-9 in CD9-HT1080 cells is sufficient to suppress the invasive phenotype. CD9-HT1080 cells were transiently transfected with short-interfering RNA directed to MMP-9 (CD9+ MMP-9 siRNA). Likewise, Mock- and CD9- HT1080 cells were transfected with a scrambled siRNA as a negative control (Mock+Ctr siRNA or CD9+ Ctr siRNA). ( A ) qRT-PCR analysis was used to measure changes in MMP-9 mRNA levels among Ctr and MMP-9 siRNA transfected cells. ( B ) Changes in release of MMP-9 (ng/ml) in to the cleared culture supernatant were examined using a MMP-9 specific ELISA kit. ( C ) Pro-MMP-9 levels in the supernatants of transiently transfected cells was measured using gelatin zymography and ( D ) quantified by densitometry. ( E ) A representative image of cells invading though matrigel after transfection with siRNA. ( F ) Percent cell invasion through matrigel-coated inserts after 20 hours was quantified and the results were normalized to Mock+Ctr siRNA treated cells *, p

    Article Snippet: A rabbit polyclonal antibody specific for the first extracellular loop of CD9 (Rap2) was also generated in our laboratory and previously reported .Anti-CD63 and anti-CD151 antibodies were purchased from BD Pharmingen (San Diego, CA).

    Techniques: Transfection, Small Interfering RNA, Negative Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Zymography

    Overexpression of CD9 in HT1080 cells does not alter the expression of other tetraspanins. ( A ) The cell surface expression of CD9 on wild-type HT1080 cells was assessed using flow cytometric analysis after binding a monoclonal antibody specific for CD9 (mAb7, shaded histogram). A non-specific isotype-matched antibody (IgG) was used as a negative control (unshaded histogram). ( B, C ) Total RNA was collected from Mock- and CD9-HT1080 cells, reverse transcribed to cDNA, and probed using primers specific for integral tetraspanins using qRT-PCR. The fold change in mRNA expression of tetraspanins was calculated from resulting cycle threshold values normalized to cyclophilin D. ( D ) Flow cytometry was used to assess the cell surface expression of key tetraspanins on the cell surface of Mock- (left panels) and CD9- (right panels) HT1080 cells **, p

    Journal: PLoS ONE

    Article Title: Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

    doi: 10.1371/journal.pone.0067766

    Figure Lengend Snippet: Overexpression of CD9 in HT1080 cells does not alter the expression of other tetraspanins. ( A ) The cell surface expression of CD9 on wild-type HT1080 cells was assessed using flow cytometric analysis after binding a monoclonal antibody specific for CD9 (mAb7, shaded histogram). A non-specific isotype-matched antibody (IgG) was used as a negative control (unshaded histogram). ( B, C ) Total RNA was collected from Mock- and CD9-HT1080 cells, reverse transcribed to cDNA, and probed using primers specific for integral tetraspanins using qRT-PCR. The fold change in mRNA expression of tetraspanins was calculated from resulting cycle threshold values normalized to cyclophilin D. ( D ) Flow cytometry was used to assess the cell surface expression of key tetraspanins on the cell surface of Mock- (left panels) and CD9- (right panels) HT1080 cells **, p

    Article Snippet: A rabbit polyclonal antibody specific for the first extracellular loop of CD9 (Rap2) was also generated in our laboratory and previously reported .Anti-CD63 and anti-CD151 antibodies were purchased from BD Pharmingen (San Diego, CA).

    Techniques: Over Expression, Expressing, Flow Cytometry, Binding Assay, Negative Control, Quantitative RT-PCR, Cytometry

    Characterization of the second extracellular loop (EC2) deletion mutant of CD9 in HT1080 cells. Upon transfection with the EC2 deletion mutant in HT1080 cells, mRNA expression was measured using primer pairs specific for the nucleotides coding the EC2 ( A ) and TM1 ( B ) regions of CD9. ( C ) Flow cytometric analysis of Mock-, CD9-, and Δ6-HT1080 cells is indicated by each row, and each column indicates CD9 EC2 (mAb7), CD9 TM1 (Rap2), or CD151 binding (shaded histograms) **, p

    Journal: PLoS ONE

    Article Title: Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

    doi: 10.1371/journal.pone.0067766

    Figure Lengend Snippet: Characterization of the second extracellular loop (EC2) deletion mutant of CD9 in HT1080 cells. Upon transfection with the EC2 deletion mutant in HT1080 cells, mRNA expression was measured using primer pairs specific for the nucleotides coding the EC2 ( A ) and TM1 ( B ) regions of CD9. ( C ) Flow cytometric analysis of Mock-, CD9-, and Δ6-HT1080 cells is indicated by each row, and each column indicates CD9 EC2 (mAb7), CD9 TM1 (Rap2), or CD151 binding (shaded histograms) **, p

    Article Snippet: A rabbit polyclonal antibody specific for the first extracellular loop of CD9 (Rap2) was also generated in our laboratory and previously reported .Anti-CD63 and anti-CD151 antibodies were purchased from BD Pharmingen (San Diego, CA).

    Techniques: Mutagenesis, Transfection, Expressing, Flow Cytometry, Binding Assay

    The invasive phenotype of HT1080 cells is increased upon CD9 overexpression. A matrigel invasion assay was used to assess the invasive properties of Mock- and CD9-HT1080 cells. Cells were allowed to invade through matrigel and adhere to the bottom of a cell culture insert as detailed in the Materials and methods section. ( A ) A representative image of stained cells after 20 hours of invasion though matrigel. ( B ) Invasion through matrigel-coated inserts was compared to cells that invaded through uncoated inserts and results shown are normalized to Mock-HT1080 cell invasion *, p

    Journal: PLoS ONE

    Article Title: Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

    doi: 10.1371/journal.pone.0067766

    Figure Lengend Snippet: The invasive phenotype of HT1080 cells is increased upon CD9 overexpression. A matrigel invasion assay was used to assess the invasive properties of Mock- and CD9-HT1080 cells. Cells were allowed to invade through matrigel and adhere to the bottom of a cell culture insert as detailed in the Materials and methods section. ( A ) A representative image of stained cells after 20 hours of invasion though matrigel. ( B ) Invasion through matrigel-coated inserts was compared to cells that invaded through uncoated inserts and results shown are normalized to Mock-HT1080 cell invasion *, p

    Article Snippet: A rabbit polyclonal antibody specific for the first extracellular loop of CD9 (Rap2) was also generated in our laboratory and previously reported .Anti-CD63 and anti-CD151 antibodies were purchased from BD Pharmingen (San Diego, CA).

    Techniques: Over Expression, Invasion Assay, Cell Culture, Staining

    CD9-HT1080 cells have stable mRNA expression of integrins though α2 and β1 are reduced at the cell surface. ( A ) The fold change in mRNA expression of integrins was compared between Mock- and CD9-HT1080 cells using qRT-PCR. ( B ) Cell surface expression of the same integrin subunits (shaded histograms) was evaluated by flow cytometry. A non-specific isotype-matched antibody (IgG) was used as a negative control (unshaded histograms).

    Journal: PLoS ONE

    Article Title: Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

    doi: 10.1371/journal.pone.0067766

    Figure Lengend Snippet: CD9-HT1080 cells have stable mRNA expression of integrins though α2 and β1 are reduced at the cell surface. ( A ) The fold change in mRNA expression of integrins was compared between Mock- and CD9-HT1080 cells using qRT-PCR. ( B ) Cell surface expression of the same integrin subunits (shaded histograms) was evaluated by flow cytometry. A non-specific isotype-matched antibody (IgG) was used as a negative control (unshaded histograms).

    Article Snippet: A rabbit polyclonal antibody specific for the first extracellular loop of CD9 (Rap2) was also generated in our laboratory and previously reported .Anti-CD63 and anti-CD151 antibodies were purchased from BD Pharmingen (San Diego, CA).

    Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Negative Control

    Anti-CD9 mAb inhibits chemotaxis toward Ag and induces tyrosine phosphorylation of NTAL by different mechanism. A, IgE-sensitized BMMCs were pretreated with the indicated concentrations of anti-CD9 mAb 2H9 for 15 min and their chemotactic response toward

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Anti-CD9 mAb inhibits chemotaxis toward Ag and induces tyrosine phosphorylation of NTAL by different mechanism. A, IgE-sensitized BMMCs were pretreated with the indicated concentrations of anti-CD9 mAb 2H9 for 15 min and their chemotactic response toward

    Article Snippet: Phospho-Tyr-specific mAb (PY-20) conjugated to HRP, anti-CD9 (KMC8), and anti-integrin β1 (HM β1–1) were purchased from BD Biosciences.

    Techniques: Chemotaxis Assay

    CD9 colocalizes with FcϵRI on the plasma membrane but CD9 aggregation does not interfere with early Ag-induced activation events. A and D , BMMCs derived from Balb/c mice were prefixed with paraformaldehyde and then labeled with anti-CD9 mAb 2H9

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: CD9 colocalizes with FcϵRI on the plasma membrane but CD9 aggregation does not interfere with early Ag-induced activation events. A and D , BMMCs derived from Balb/c mice were prefixed with paraformaldehyde and then labeled with anti-CD9 mAb 2H9

    Article Snippet: Phospho-Tyr-specific mAb (PY-20) conjugated to HRP, anti-CD9 (KMC8), and anti-integrin β1 (HM β1–1) were purchased from BD Biosciences.

    Techniques: Activation Assay, Derivative Assay, Mouse Assay, Labeling

    Identification of CD9 as the target protein of 2H9 mAb. 2H9 mAb covalently bound to protein G resin by dimethylpimelimidate was used to pulldown the target Ag from postnuclear supernatant of BMMCs lysed in a lysis buffer containing 1% Triton X-100. Bound

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Identification of CD9 as the target protein of 2H9 mAb. 2H9 mAb covalently bound to protein G resin by dimethylpimelimidate was used to pulldown the target Ag from postnuclear supernatant of BMMCs lysed in a lysis buffer containing 1% Triton X-100. Bound

    Article Snippet: Phospho-Tyr-specific mAb (PY-20) conjugated to HRP, anti-CD9 (KMC8), and anti-integrin β1 (HM β1–1) were purchased from BD Biosciences.

    Techniques: Lysis

    CD9 aggregation does not interfere with β1-integrin function, but induces dephosphorylation of ERM proteins. A-D , BMMCs were pretreated or not with anti-CD9 mAb 2H9 (1 μg/ml) for 15 min and the binding anti-integrin-β1-FITC conjugate

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: CD9 aggregation does not interfere with β1-integrin function, but induces dephosphorylation of ERM proteins. A-D , BMMCs were pretreated or not with anti-CD9 mAb 2H9 (1 μg/ml) for 15 min and the binding anti-integrin-β1-FITC conjugate

    Article Snippet: Phospho-Tyr-specific mAb (PY-20) conjugated to HRP, anti-CD9 (KMC8), and anti-integrin β1 (HM β1–1) were purchased from BD Biosciences.

    Techniques: De-Phosphorylation Assay, Binding Assay

    A model of chemotaxis regulators involving CD9 and NTAL-LAT cross-talk. Tetraspanin CD9 resides in the plasma membrane ( PM ) in close proximity to β1-integrin (β1) and NTAL. CD9-specific antibody 2H9 ( Ab ) binds to CD9 and FcγR through

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: A model of chemotaxis regulators involving CD9 and NTAL-LAT cross-talk. Tetraspanin CD9 resides in the plasma membrane ( PM ) in close proximity to β1-integrin (β1) and NTAL. CD9-specific antibody 2H9 ( Ab ) binds to CD9 and FcγR through

    Article Snippet: Phospho-Tyr-specific mAb (PY-20) conjugated to HRP, anti-CD9 (KMC8), and anti-integrin β1 (HM β1–1) were purchased from BD Biosciences.

    Techniques: Chemotaxis Assay

    Different roles of LAT and NTAL in mast cell chemotaxis and cross-talk with CD9. A , BMMCs derived from Lat −/− , Ntal −/− , 2KO, and corresponding littermate ( Lat +/+ , Ntal +/+ ) control mice were sensitized overnight with TNP-specific

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Different roles of LAT and NTAL in mast cell chemotaxis and cross-talk with CD9. A , BMMCs derived from Lat −/− , Ntal −/− , 2KO, and corresponding littermate ( Lat +/+ , Ntal +/+ ) control mice were sensitized overnight with TNP-specific

    Article Snippet: Phospho-Tyr-specific mAb (PY-20) conjugated to HRP, anti-CD9 (KMC8), and anti-integrin β1 (HM β1–1) were purchased from BD Biosciences.

    Techniques: Chemotaxis Assay, Derivative Assay, Mouse Assay

    Different colocalization of CD9 with NTAL or LAT. BMMCs were prefixed in 2% paraformaldehyde and then stained with 2H9 mAb followed by secondary antibody conjugated to 12 nm of gold ( A, D, G, and J ). Alternatively, the cells were first treated with 2H9

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Different colocalization of CD9 with NTAL or LAT. BMMCs were prefixed in 2% paraformaldehyde and then stained with 2H9 mAb followed by secondary antibody conjugated to 12 nm of gold ( A, D, G, and J ). Alternatively, the cells were first treated with 2H9

    Article Snippet: Phospho-Tyr-specific mAb (PY-20) conjugated to HRP, anti-CD9 (KMC8), and anti-integrin β1 (HM β1–1) were purchased from BD Biosciences.

    Techniques: Staining

    TEM and western blotting was applied to verify the presence of EV subpopulations in both washed and unwashed pellets. TEM showing EV-characteristics, i.e. shape and size in (A) unwashed and (B) washed pellets. Immunogold labelling with anti-CD9 (clone M-L13) bound to EVs in unwashed (C) and washed (D) pellets confirming presence of CD9-positive subpopulations. (E) Small (

    Journal: Journal of Extracellular Vesicles

    Article Title: Investigation of procoagulant activity in extracellular vesicles isolated by differential ultracentrifugation

    doi: 10.1080/20013078.2018.1454777

    Figure Lengend Snippet: TEM and western blotting was applied to verify the presence of EV subpopulations in both washed and unwashed pellets. TEM showing EV-characteristics, i.e. shape and size in (A) unwashed and (B) washed pellets. Immunogold labelling with anti-CD9 (clone M-L13) bound to EVs in unwashed (C) and washed (D) pellets confirming presence of CD9-positive subpopulations. (E) Small (

    Article Snippet: After blockage, the grids were incubated in one drop of primary anti-CD9 antibody (clone M-L13, BD Pharmingen, San Jose, CA, USA) 1:50 in 0.5% ovalbumin in PBS for 30 min at 37°C.

    Techniques: Transmission Electron Microscopy, Western Blot

    CD9 is required for the gp130-mediated BMX-STAT3 signaling in GSCs. ( a , b ) Immunoblot analyses of CD9, gp130, p-BMX (p-Tyr40), BMX, p-STAT3 (p-Tyr705) and STAT3 in D456-GSCs ( a ) or T4121-GSCs ( b ) expressing shCD9 or shNT in combination with IL-6 treatment. Cells were treated with IL-6 (10 ng/ml) or control vehicle for 10 min and were collected for immunoblot analyses. CD9 disruption abolished the IL-6 induced activating phosphorylations of BMX (p-Tyr40) and STAT3 (p-Tyr705). ( c , d ) Immunoblot analyses of p-STAT3 (p-Tyr705), STAT3, Bcl-xL and c-Myc in D456-GSCs ( c ) or T4121-GSCs ( d ) expressing shCD9 or shNT. Silencing CD9 attenuated the activity of STAT3 (p-STAT3) and thus suppressed the expressions of STAT3 downstream targets Bcl-xL and c-Myc. All experiments were performed independently for three times

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: CD9 is required for the gp130-mediated BMX-STAT3 signaling in GSCs. ( a , b ) Immunoblot analyses of CD9, gp130, p-BMX (p-Tyr40), BMX, p-STAT3 (p-Tyr705) and STAT3 in D456-GSCs ( a ) or T4121-GSCs ( b ) expressing shCD9 or shNT in combination with IL-6 treatment. Cells were treated with IL-6 (10 ng/ml) or control vehicle for 10 min and were collected for immunoblot analyses. CD9 disruption abolished the IL-6 induced activating phosphorylations of BMX (p-Tyr40) and STAT3 (p-Tyr705). ( c , d ) Immunoblot analyses of p-STAT3 (p-Tyr705), STAT3, Bcl-xL and c-Myc in D456-GSCs ( c ) or T4121-GSCs ( d ) expressing shCD9 or shNT. Silencing CD9 attenuated the activity of STAT3 (p-STAT3) and thus suppressed the expressions of STAT3 downstream targets Bcl-xL and c-Myc. All experiments were performed independently for three times

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Activity Assay

    Expression of a STAT3-C compromises GSC maintenance impaired by CD9 disruption. ( a ) Immunoblot analyses of Flag-tagged STAT3-C, STAT3 and CD9 in D456 GSCs transduced with STAT3-C or vector and shCD9 or shNT control. ( b – d ) The representative images ( b ) and quantification of sphere numbers ( c ) and sphere diameters ( d ) of D456 GSCs expressing shCD9 and/or STAT3-C. Scale bar=100 μ m. STAT3-C compromised the inhibitory effect of CD9 disruption on GSC tumorsphere formation. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Expression of a STAT3-C compromises GSC maintenance impaired by CD9 disruption. ( a ) Immunoblot analyses of Flag-tagged STAT3-C, STAT3 and CD9 in D456 GSCs transduced with STAT3-C or vector and shCD9 or shNT control. ( b – d ) The representative images ( b ) and quantification of sphere numbers ( c ) and sphere diameters ( d ) of D456 GSCs expressing shCD9 and/or STAT3-C. Scale bar=100 μ m. STAT3-C compromised the inhibitory effect of CD9 disruption on GSC tumorsphere formation. ** P

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Transduction, Plasmid Preparation

    CD9 interacts with gp130 to mediate its function in GSCs. ( a ) MS analysis identified gp130 as the key interacting protein of CD9. CD9 was immunoprecipitated from T4121 GSCs expressing CD9-Flag using anti-Flag-conjugated beads. Gp130 was identified through MS analysis by peptides covering the gp130 protein sequence. A representative detected peptide (TNHFTIPK) of gp130 was shown. ( b ) Co-immunoprecipitation of gp130 with CD9 using anti-Flag-conjugated beads in D456 and T4121 GSCs transduced with CD9-Flag or vector control. ( c ) Representative immunofluorescent images of CD9 (in green) and gp130 (in red) in D456 and T4121 GSCs. Co-localization of CD9 and gp130 was detected in GSCs. Scale bar represents 10 μ m. ( d ) Quantification of co-localization rate of CD9 and gp130 in c . The co-localization rate of CD9 with gp130 was determined with five randomly selected images using Leica LAS AF Lite software. ( e ) Immunoblot analysis of gp130 in GSCs expressing shgp130 or shNT. Disrupting gp130 by shRNA effectively reduced gp130 expression. ( f ) Cell proliferation assay of GSCs expressing shgp130 or shNT. Gp130 disruption significantly suppressed GSC proliferation. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: CD9 interacts with gp130 to mediate its function in GSCs. ( a ) MS analysis identified gp130 as the key interacting protein of CD9. CD9 was immunoprecipitated from T4121 GSCs expressing CD9-Flag using anti-Flag-conjugated beads. Gp130 was identified through MS analysis by peptides covering the gp130 protein sequence. A representative detected peptide (TNHFTIPK) of gp130 was shown. ( b ) Co-immunoprecipitation of gp130 with CD9 using anti-Flag-conjugated beads in D456 and T4121 GSCs transduced with CD9-Flag or vector control. ( c ) Representative immunofluorescent images of CD9 (in green) and gp130 (in red) in D456 and T4121 GSCs. Co-localization of CD9 and gp130 was detected in GSCs. Scale bar represents 10 μ m. ( d ) Quantification of co-localization rate of CD9 and gp130 in c . The co-localization rate of CD9 with gp130 was determined with five randomly selected images using Leica LAS AF Lite software. ( e ) Immunoblot analysis of gp130 in GSCs expressing shgp130 or shNT. Disrupting gp130 by shRNA effectively reduced gp130 expression. ( f ) Cell proliferation assay of GSCs expressing shgp130 or shNT. Gp130 disruption significantly suppressed GSC proliferation. ** P

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Mass Spectrometry, Immunoprecipitation, Expressing, Sequencing, Transduction, Plasmid Preparation, Software, shRNA, Proliferation Assay

    Expression of the STAT-C restores GSC-driven tumor propagation impaired by CD9 disruption. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 and/or STAT3-C at day 17 and day 24 after tumor implantation. Data are shown as mean±S.E.M. ( b ). ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Expression of the STAT-C restores GSC-driven tumor propagation impaired by CD9 disruption. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 and/or STAT3-C at day 17 and day 24 after tumor implantation. Data are shown as mean±S.E.M. ( b ). ** P

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Derivative Assay, Tumor Implantation

    A schematic diagram shows that CD9 binds to gp130 and stabilizes gp130 by blocking its ubiquitin-dependent lysosomal degradation to promote the BMX-mediated STAT3 activation in GSCs. The binding of CD9 to gp130 prevents gp130 from ubiquitination and lysosomal degradation, thus facilitates the signal transduction of the IL-6-gp130-BMX-STAT3 signaling pathway in GSCs to maintain GSC self-renewal and tumorigenic potential

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: A schematic diagram shows that CD9 binds to gp130 and stabilizes gp130 by blocking its ubiquitin-dependent lysosomal degradation to promote the BMX-mediated STAT3 activation in GSCs. The binding of CD9 to gp130 prevents gp130 from ubiquitination and lysosomal degradation, thus facilitates the signal transduction of the IL-6-gp130-BMX-STAT3 signaling pathway in GSCs to maintain GSC self-renewal and tumorigenic potential

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Blocking Assay, Activation Assay, Binding Assay, Transduction

    CD9 stabilizes gp130 by preventing the ubiquitin-dependent lysosomal degradation. ( a , b ) qRT-PCR analysis of CD9 ( a ) and IL6ST (gene encoding for gp130) ( b ) in D456 and T4121 GSCs expressing shCD9 or shNT. Silencing CD9 did not affect mRNA level of gp130 in GSCs. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: CD9 stabilizes gp130 by preventing the ubiquitin-dependent lysosomal degradation. ( a , b ) qRT-PCR analysis of CD9 ( a ) and IL6ST (gene encoding for gp130) ( b ) in D456 and T4121 GSCs expressing shCD9 or shNT. Silencing CD9 did not affect mRNA level of gp130 in GSCs. ** P

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Quantitative RT-PCR, Expressing

    Tetraspanin CD9 is preferentially expressed in GSCs and is essential for the GSC maintenance. ( a ) The expression heatmap of tetraspanins in GSC lines ( n =12) relative to CGCs ( n =32) from the GEO profiles (GEO: GDS3885). Four candidates, including CD9 , TSPAN7 , TSPAN11 and TSPAN33 were significantly upregulated in GSCs relative to CGCs. Data were visualized using Cluster/Java Treeview. ( b ) Immunoblot analysis showing the preferential expressions of CD9 and the GSC marker SOX2 in GSCs ( n =6) relative to the matched NSTCs ( n =6) isolated from human GBMs. ( c ) Immunofluorescent staining of CD9 (in green) and the GSC marker SOX2 (in red, upper panel), OLIG2 (in red, middle panel) or CD133 (in red, lower panel) in GSC tumorspheres. Scale bar represents 25 μ m. ( d ) Immunoblot analyses of CD9, the GSC marker SOX2 and the neuronal differentiation marker MAP2 during the serum-induced differentiation of GSCs. The levels of CD9 and the GSC marker SOX2 decreased, while the expression of the differentiation marker MAP2 concomitantly increased over a 7-day period. ( e ) In vitro limiting dilution analyses of the secondary tumorsphere formations of GSCs expressing shCD9 (shCD9-1 and -2) or non-targeting shRNA (shNT, control). Disrupting CD9 expression attenuated the self-renewal capacity of GSCs. ** P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Tetraspanin CD9 is preferentially expressed in GSCs and is essential for the GSC maintenance. ( a ) The expression heatmap of tetraspanins in GSC lines ( n =12) relative to CGCs ( n =32) from the GEO profiles (GEO: GDS3885). Four candidates, including CD9 , TSPAN7 , TSPAN11 and TSPAN33 were significantly upregulated in GSCs relative to CGCs. Data were visualized using Cluster/Java Treeview. ( b ) Immunoblot analysis showing the preferential expressions of CD9 and the GSC marker SOX2 in GSCs ( n =6) relative to the matched NSTCs ( n =6) isolated from human GBMs. ( c ) Immunofluorescent staining of CD9 (in green) and the GSC marker SOX2 (in red, upper panel), OLIG2 (in red, middle panel) or CD133 (in red, lower panel) in GSC tumorspheres. Scale bar represents 25 μ m. ( d ) Immunoblot analyses of CD9, the GSC marker SOX2 and the neuronal differentiation marker MAP2 during the serum-induced differentiation of GSCs. The levels of CD9 and the GSC marker SOX2 decreased, while the expression of the differentiation marker MAP2 concomitantly increased over a 7-day period. ( e ) In vitro limiting dilution analyses of the secondary tumorsphere formations of GSCs expressing shCD9 (shCD9-1 and -2) or non-targeting shRNA (shNT, control). Disrupting CD9 expression attenuated the self-renewal capacity of GSCs. ** P

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Expressing, Marker, Isolation, Staining, In Vitro, shRNA

    Disrupting CD9 inhibits GSC-driven tumor growth and STAT3 activation in vivo. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 or shNT at day 14 and day 28 after tumor implantation. Silencing CD9 markedly suppressed GSC-driven tumor growth. * P

    Journal: Cell Death and Differentiation

    Article Title: Tetraspanin CD9 stabilizes gp130 by preventing its ubiquitin-dependent lysosomal degradation to promote STAT3 activation in glioma stem cells

    doi: 10.1038/cdd.2016.110

    Figure Lengend Snippet: Disrupting CD9 inhibits GSC-driven tumor growth and STAT3 activation in vivo. ( a , b ) Representative bioluminescent images ( a ) and the quantification ( b ) of xenografts derived from T4121 GSCs expressing shCD9 or shNT at day 14 and day 28 after tumor implantation. Silencing CD9 markedly suppressed GSC-driven tumor growth. * P

    Article Snippet: Immunoblot analyses were performed as previously described., The primary antibodies used in this study include the following: anti-CD9 (sc-13118, 1:1000, Santa Cruz); anti-gp130 (sc-655, 1:1000, Santa Cruz); anti-c-Myc (sc-40, 1:1000, Santa Cruz); anti-Cbl (sc-1651, 1:500, Santa Cruz); anti-p-STAT3 (p-Tyr705, #9131, 1:1000, Cell Signaling, Danvers, MA, USA); anti-STAT3 (#9139, 1:2000, Cell Signaling); anti-Bcl-xL (#2764, 1:500, Cell Signaling); anti-p-BMX (p-Tyr40, #3211, 1:1000, Cell Signaling); anti-BMX (ab59360, 1:1000, Abcam, Cambridge, UK); anti-Ubiquitin (646301, 1:500, Biolegend, San Diego, CA, USA); anti-GFAP (644702, 1:1000, Biolegend); anti-MAP2 (SMI-52 R, 1:1000, Covance, Princeton, NJ, USA); anti-Flag (637301, 1:1000, Biolegend); anti-GAPDH (#2118, 1:5000, Cell Signaling); and anti-tubulin (T9026, 1:10 000, Sigma).

    Techniques: Activation Assay, In Vivo, Derivative Assay, Expressing, Tumor Implantation

    Anti-CD9 mAb inhibits chemotaxis toward Ag and induces tyrosine phosphorylation of NTAL by different mechanism. A, IgE-sensitized BMMCs were pretreated with the indicated concentrations of anti-CD9 mAb 2H9 for 15 min and their chemotactic response toward

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Anti-CD9 mAb inhibits chemotaxis toward Ag and induces tyrosine phosphorylation of NTAL by different mechanism. A, IgE-sensitized BMMCs were pretreated with the indicated concentrations of anti-CD9 mAb 2H9 for 15 min and their chemotactic response toward

    Article Snippet: Specificity of the 2H9 antibody was verified by immunoprecipitation followed by mass spectrometry analysis as described ( ) and by cross-immunoprecipitation using commercially available anti-CD9 antibody (KMC8.8, Santa Cruz Biotechnology, Inc.).

    Techniques: Chemotaxis Assay

    CD9 colocalizes with FcϵRI on the plasma membrane but CD9 aggregation does not interfere with early Ag-induced activation events. A and D , BMMCs derived from Balb/c mice were prefixed with paraformaldehyde and then labeled with anti-CD9 mAb 2H9

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: CD9 colocalizes with FcϵRI on the plasma membrane but CD9 aggregation does not interfere with early Ag-induced activation events. A and D , BMMCs derived from Balb/c mice were prefixed with paraformaldehyde and then labeled with anti-CD9 mAb 2H9

    Article Snippet: Specificity of the 2H9 antibody was verified by immunoprecipitation followed by mass spectrometry analysis as described ( ) and by cross-immunoprecipitation using commercially available anti-CD9 antibody (KMC8.8, Santa Cruz Biotechnology, Inc.).

    Techniques: Activation Assay, Derivative Assay, Mouse Assay, Labeling

    Identification of CD9 as the target protein of 2H9 mAb. 2H9 mAb covalently bound to protein G resin by dimethylpimelimidate was used to pulldown the target Ag from postnuclear supernatant of BMMCs lysed in a lysis buffer containing 1% Triton X-100. Bound

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Identification of CD9 as the target protein of 2H9 mAb. 2H9 mAb covalently bound to protein G resin by dimethylpimelimidate was used to pulldown the target Ag from postnuclear supernatant of BMMCs lysed in a lysis buffer containing 1% Triton X-100. Bound

    Article Snippet: Specificity of the 2H9 antibody was verified by immunoprecipitation followed by mass spectrometry analysis as described ( ) and by cross-immunoprecipitation using commercially available anti-CD9 antibody (KMC8.8, Santa Cruz Biotechnology, Inc.).

    Techniques: Lysis

    CD9 aggregation does not interfere with β1-integrin function, but induces dephosphorylation of ERM proteins. A-D , BMMCs were pretreated or not with anti-CD9 mAb 2H9 (1 μg/ml) for 15 min and the binding anti-integrin-β1-FITC conjugate

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: CD9 aggregation does not interfere with β1-integrin function, but induces dephosphorylation of ERM proteins. A-D , BMMCs were pretreated or not with anti-CD9 mAb 2H9 (1 μg/ml) for 15 min and the binding anti-integrin-β1-FITC conjugate

    Article Snippet: Specificity of the 2H9 antibody was verified by immunoprecipitation followed by mass spectrometry analysis as described ( ) and by cross-immunoprecipitation using commercially available anti-CD9 antibody (KMC8.8, Santa Cruz Biotechnology, Inc.).

    Techniques: De-Phosphorylation Assay, Binding Assay

    A model of chemotaxis regulators involving CD9 and NTAL-LAT cross-talk. Tetraspanin CD9 resides in the plasma membrane ( PM ) in close proximity to β1-integrin (β1) and NTAL. CD9-specific antibody 2H9 ( Ab ) binds to CD9 and FcγR through

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: A model of chemotaxis regulators involving CD9 and NTAL-LAT cross-talk. Tetraspanin CD9 resides in the plasma membrane ( PM ) in close proximity to β1-integrin (β1) and NTAL. CD9-specific antibody 2H9 ( Ab ) binds to CD9 and FcγR through

    Article Snippet: Specificity of the 2H9 antibody was verified by immunoprecipitation followed by mass spectrometry analysis as described ( ) and by cross-immunoprecipitation using commercially available anti-CD9 antibody (KMC8.8, Santa Cruz Biotechnology, Inc.).

    Techniques: Chemotaxis Assay

    Different roles of LAT and NTAL in mast cell chemotaxis and cross-talk with CD9. A , BMMCs derived from Lat −/− , Ntal −/− , 2KO, and corresponding littermate ( Lat +/+ , Ntal +/+ ) control mice were sensitized overnight with TNP-specific

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Different roles of LAT and NTAL in mast cell chemotaxis and cross-talk with CD9. A , BMMCs derived from Lat −/− , Ntal −/− , 2KO, and corresponding littermate ( Lat +/+ , Ntal +/+ ) control mice were sensitized overnight with TNP-specific

    Article Snippet: Specificity of the 2H9 antibody was verified by immunoprecipitation followed by mass spectrometry analysis as described ( ) and by cross-immunoprecipitation using commercially available anti-CD9 antibody (KMC8.8, Santa Cruz Biotechnology, Inc.).

    Techniques: Chemotaxis Assay, Derivative Assay, Mouse Assay

    Different colocalization of CD9 with NTAL or LAT. BMMCs were prefixed in 2% paraformaldehyde and then stained with 2H9 mAb followed by secondary antibody conjugated to 12 nm of gold ( A, D, G, and J ). Alternatively, the cells were first treated with 2H9

    Journal: The Journal of Biological Chemistry

    Article Title: Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis *

    doi: 10.1074/jbc.M112.449231

    Figure Lengend Snippet: Different colocalization of CD9 with NTAL or LAT. BMMCs were prefixed in 2% paraformaldehyde and then stained with 2H9 mAb followed by secondary antibody conjugated to 12 nm of gold ( A, D, G, and J ). Alternatively, the cells were first treated with 2H9

    Article Snippet: Specificity of the 2H9 antibody was verified by immunoprecipitation followed by mass spectrometry analysis as described ( ) and by cross-immunoprecipitation using commercially available anti-CD9 antibody (KMC8.8, Santa Cruz Biotechnology, Inc.).

    Techniques: Staining

    The molecular characteristics of CAR-T and CD19 + B cells. (A–B) Expression levels of the selected genes in CAR-T and CD19 + B cells before and after treatment were detected by RNA-seq. Left panel: the gene expression details of CAR-T or CD19 + B cells in pretreatment and post-treatment samples; right panel: the fold change in the expression of selected genes in post-treatment samples derived CAR-T or CD19 + B cells (relative to the pretreatment value). (C) The CD19 + B cells isolated from tumor tissues of relapsed patient exhibited a missense mutation in exon 3 (P. 163 R > L/C.488 G > T). (D) The autologous CAR (FMC63)-T cells cocultured with CD19 + B cells were derived from bone marrow before treatment and from tumor tissues after relapse at a ratio of 10:1. The CAR-T cells are potentiated to eradicate normal CD19 + cells, but not the mutated target cells. (E) The FMC63 and 21D4 CAR-T cells were incubated with target cells derived from tumor tissues. *p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy

    doi: 10.1136/jitc-2020-001150

    Figure Lengend Snippet: The molecular characteristics of CAR-T and CD19 + B cells. (A–B) Expression levels of the selected genes in CAR-T and CD19 + B cells before and after treatment were detected by RNA-seq. Left panel: the gene expression details of CAR-T or CD19 + B cells in pretreatment and post-treatment samples; right panel: the fold change in the expression of selected genes in post-treatment samples derived CAR-T or CD19 + B cells (relative to the pretreatment value). (C) The CD19 + B cells isolated from tumor tissues of relapsed patient exhibited a missense mutation in exon 3 (P. 163 R > L/C.488 G > T). (D) The autologous CAR (FMC63)-T cells cocultured with CD19 + B cells were derived from bone marrow before treatment and from tumor tissues after relapse at a ratio of 10:1. The CAR-T cells are potentiated to eradicate normal CD19 + cells, but not the mutated target cells. (E) The FMC63 and 21D4 CAR-T cells were incubated with target cells derived from tumor tissues. *p

    Article Snippet: Cells were then incubated with anti-CD3-PE-cy7 (clone UCHT1; BD Pharmingen), CD19 (20-291) protein-FITC (ACRO Biosystems), or anti-CD19-PE (clone HIB19; BioLegend) antibodies for 30 min. Flow cytometry analysis was performed on BD FACS CantoII (BD, USA).

    Techniques: Expressing, RNA Sequencing Assay, Derivative Assay, Isolation, Mutagenesis, Incubation

    21D4 CAR-T cells exhibit a potent cytotoxicity against mutated CD19 + cells derived from patients with no response (NR). (A) Quantitative real-time-PCR analysis of CAR-T cells in peripheral blood. (B) Lymphoma patients with NR had a point mutation in exon 3 (P. 174 L > V/C.520 C > G) of CD19 . (C) FMC63 or 21D4 CAR-T cells were cocultured with mutated CD19 + B cells derived from patients at E:T ratio of 5:1 for 6 hours. Lysis of CD19 + cells was detected and 21D4 CAR-T cells exhibited higher lysis ability than FMC63 CAR-T cells in the patients with NR. **p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy

    doi: 10.1136/jitc-2020-001150

    Figure Lengend Snippet: 21D4 CAR-T cells exhibit a potent cytotoxicity against mutated CD19 + cells derived from patients with no response (NR). (A) Quantitative real-time-PCR analysis of CAR-T cells in peripheral blood. (B) Lymphoma patients with NR had a point mutation in exon 3 (P. 174 L > V/C.520 C > G) of CD19 . (C) FMC63 or 21D4 CAR-T cells were cocultured with mutated CD19 + B cells derived from patients at E:T ratio of 5:1 for 6 hours. Lysis of CD19 + cells was detected and 21D4 CAR-T cells exhibited higher lysis ability than FMC63 CAR-T cells in the patients with NR. **p

    Article Snippet: Cells were then incubated with anti-CD3-PE-cy7 (clone UCHT1; BD Pharmingen), CD19 (20-291) protein-FITC (ACRO Biosystems), or anti-CD19-PE (clone HIB19; BioLegend) antibodies for 30 min. Flow cytometry analysis was performed on BD FACS CantoII (BD, USA).

    Techniques: Derivative Assay, Real-time Polymerase Chain Reaction, Mutagenesis, Lysis

    Treatment regimen and kinetics of CD19 CAR-T cells in a patient with high-grade B cell lymphoma. (A) Timeline of the CAR-T protocol conducted for one patient with lymphoma. (B) The positron emission tomography-CT scan images of this patient before treatment, and on days 60, 180, and 356 post-CD19 CAR-T cell infusion are shown. (C) The percentage of CD19 + cells derived from peripheral blood mononuclear cells was detected by flow cytometry analysis. (D) Time course analysis of expression levels of CD19 CAR transgene by quantitative real-time-PCR. (E) The fractions of CAR-T and CD19 + B cells in the tissue and bone marrow were detected by flow cytometry on day 180. CAR, chimeric antigen receptor; CR, complete response; CTX, cyclophosphamide; FLU, fludarabine; PD1, programmed cell death protein 1; SSC-A, side scatter-A.

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy

    doi: 10.1136/jitc-2020-001150

    Figure Lengend Snippet: Treatment regimen and kinetics of CD19 CAR-T cells in a patient with high-grade B cell lymphoma. (A) Timeline of the CAR-T protocol conducted for one patient with lymphoma. (B) The positron emission tomography-CT scan images of this patient before treatment, and on days 60, 180, and 356 post-CD19 CAR-T cell infusion are shown. (C) The percentage of CD19 + cells derived from peripheral blood mononuclear cells was detected by flow cytometry analysis. (D) Time course analysis of expression levels of CD19 CAR transgene by quantitative real-time-PCR. (E) The fractions of CAR-T and CD19 + B cells in the tissue and bone marrow were detected by flow cytometry on day 180. CAR, chimeric antigen receptor; CR, complete response; CTX, cyclophosphamide; FLU, fludarabine; PD1, programmed cell death protein 1; SSC-A, side scatter-A.

    Article Snippet: Cells were then incubated with anti-CD3-PE-cy7 (clone UCHT1; BD Pharmingen), CD19 (20-291) protein-FITC (ACRO Biosystems), or anti-CD19-PE (clone HIB19; BioLegend) antibodies for 30 min. Flow cytometry analysis was performed on BD FACS CantoII (BD, USA).

    Techniques: Positron Emission Tomography, Computed Tomography, Derivative Assay, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction

    21D4 CAR-T cells show enhanced effector-specific function following stimulation by loss of exon or mutated CD19 + targeted cells in vitro. (A) FMC63 and 21D4 CAR-Τ cells were incubated with wild type or mutant (exon3, p.163). (R-L) CD19-H322 and A549 cells for 24 hours. Then the viabilities of target cells were evaluated based on bioluminescent imaging intensities. (B). Tumor cells were incubated with CAR-T cells for 6 hours. Then the tumor cells were collected and stained with Annexin-V/PI to determine specific lysis. (C) The CD19 + cells with deleted exon 1, 2, 3, or 4 were collected and co-cultured with CAR-T cells for 24 hours. Then the lysis of target cells was evaluated based on BLI intensities. (D) The specific lysis of CD19 + tumor cells with deleted exon was detected by flow cytometry. *p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy

    doi: 10.1136/jitc-2020-001150

    Figure Lengend Snippet: 21D4 CAR-T cells show enhanced effector-specific function following stimulation by loss of exon or mutated CD19 + targeted cells in vitro. (A) FMC63 and 21D4 CAR-Τ cells were incubated with wild type or mutant (exon3, p.163). (R-L) CD19-H322 and A549 cells for 24 hours. Then the viabilities of target cells were evaluated based on bioluminescent imaging intensities. (B). Tumor cells were incubated with CAR-T cells for 6 hours. Then the tumor cells were collected and stained with Annexin-V/PI to determine specific lysis. (C) The CD19 + cells with deleted exon 1, 2, 3, or 4 were collected and co-cultured with CAR-T cells for 24 hours. Then the lysis of target cells was evaluated based on BLI intensities. (D) The specific lysis of CD19 + tumor cells with deleted exon was detected by flow cytometry. *p

    Article Snippet: Cells were then incubated with anti-CD3-PE-cy7 (clone UCHT1; BD Pharmingen), CD19 (20-291) protein-FITC (ACRO Biosystems), or anti-CD19-PE (clone HIB19; BioLegend) antibodies for 30 min. Flow cytometry analysis was performed on BD FACS CantoII (BD, USA).

    Techniques: In Vitro, Incubation, Mutagenesis, Imaging, Staining, Lysis, Cell Culture, Flow Cytometry

    Antitumor ability of CD19 CAR-T cells in vivo. (A–C) The efficacy of CD19 CAR-T cells was examined using a mouse model. Briefly, 1×10 6 luciferase-transgenic Raji cells were inoculated into SCID-beige mice via tail on day 7. The 1×10 6 wild type or mutated CD19 positive H322/Luc cells were subcutaneously injected into the mice. On day 0, 1×10 7 non-infected T cells or FMC63 and 21D4 CAR-Τ cells were injected intravenously (intravenous). Then, tumor growth was monitored via bioluminescent imaging test at indicated time points. The tumor growth was controlled by both CAR-T cells in wild type CD19 model. Moreover, 21D4 CAR-T cells showed better antitumor function than that of FMC CAR-T cells in mutated CD19 model. The expression values of the photon flux were transformed into log2 values for further analysis. (D) The survival curves were analyzed using the log-rank test, and the statistical result showed that a significant difference between FMC63 and 21D4 CAR-T cells treated H322-mCD19 mice. *p

    Journal: Journal for Immunotherapy of Cancer

    Article Title: Point mutation in CD19 facilitates immune escape of B cell lymphoma from CAR-T cell therapy

    doi: 10.1136/jitc-2020-001150

    Figure Lengend Snippet: Antitumor ability of CD19 CAR-T cells in vivo. (A–C) The efficacy of CD19 CAR-T cells was examined using a mouse model. Briefly, 1×10 6 luciferase-transgenic Raji cells were inoculated into SCID-beige mice via tail on day 7. The 1×10 6 wild type or mutated CD19 positive H322/Luc cells were subcutaneously injected into the mice. On day 0, 1×10 7 non-infected T cells or FMC63 and 21D4 CAR-Τ cells were injected intravenously (intravenous). Then, tumor growth was monitored via bioluminescent imaging test at indicated time points. The tumor growth was controlled by both CAR-T cells in wild type CD19 model. Moreover, 21D4 CAR-T cells showed better antitumor function than that of FMC CAR-T cells in mutated CD19 model. The expression values of the photon flux were transformed into log2 values for further analysis. (D) The survival curves were analyzed using the log-rank test, and the statistical result showed that a significant difference between FMC63 and 21D4 CAR-T cells treated H322-mCD19 mice. *p

    Article Snippet: Cells were then incubated with anti-CD3-PE-cy7 (clone UCHT1; BD Pharmingen), CD19 (20-291) protein-FITC (ACRO Biosystems), or anti-CD19-PE (clone HIB19; BioLegend) antibodies for 30 min. Flow cytometry analysis was performed on BD FACS CantoII (BD, USA).

    Techniques: In Vivo, Luciferase, Transgenic Assay, Mouse Assay, Injection, Infection, Imaging, Expressing, Transformation Assay

    Western blot analysis. Western blot was performed on cExo and ncExo samples, both from exosome-fractions (fractions 7–9) and from a non-exosome-fraction (fraction 14). Normal human keratinocyte lysate was used as control. (a) CD9 was positive in all exosome-samples and in the lysate, but negative in the non-exosome-fraction. (b) Flotillin was positive in all exosome-fractions and lysate, but with varying signal strength, and no signal was detected in the non-exosome-fraction. (c) Annexin V was negative in all fractions, but positive in the lysate. (d) EpCAM was negative in all samples (expected at 40 kDa, dotted box). (e) HSP70, heat shock protein 70, was negative in all samples except for cell lysate control. (f) GRP94, an endoplasmic reticulum protein that commonly contaminates vesicle isolates, was also negative in all samples except for cell lysate control. d3, d4 and d5 refer to donors 3, 4 and 5.

    Journal: Journal of Extracellular Vesicles

    Article Title: Exosomes derived from clinical-grade oral mucosal epithelial cell sheets promote wound healing

    doi: 10.1080/20013078.2019.1565264

    Figure Lengend Snippet: Western blot analysis. Western blot was performed on cExo and ncExo samples, both from exosome-fractions (fractions 7–9) and from a non-exosome-fraction (fraction 14). Normal human keratinocyte lysate was used as control. (a) CD9 was positive in all exosome-samples and in the lysate, but negative in the non-exosome-fraction. (b) Flotillin was positive in all exosome-fractions and lysate, but with varying signal strength, and no signal was detected in the non-exosome-fraction. (c) Annexin V was negative in all fractions, but positive in the lysate. (d) EpCAM was negative in all samples (expected at 40 kDa, dotted box). (e) HSP70, heat shock protein 70, was negative in all samples except for cell lysate control. (f) GRP94, an endoplasmic reticulum protein that commonly contaminates vesicle isolates, was also negative in all samples except for cell lysate control. d3, d4 and d5 refer to donors 3, 4 and 5.

    Article Snippet: The antibodies and concentration used were: CD9 (#13174, Cell Signaling Technologies, 1:1000, ref 02/2017, lot 1), Annexin 5 (#8555, Cell Signaling Technologies, 1:1000, ref 10/2016, lot 1), Flotillin (#18634, Cell Signaling Technologies, 1:1000, ref 10/2016 lot 1), GRP94 (#2104S, Cell Signaling Technologies, 1:1000, ref 04/2017, lot 2), HSP70 (#4876T, Cell Signaling Technologies, 1:1000, ref 10/2016, lot 2), EpCAM (#2626T, Cell Signaling Technologies, 1:1000, ref 10/2016, lot 1) and Anti-rabbit IgG, HRP-linked (#7074, Cell Signaling Technologies, 1:5000, ref 09/2016, lot 26).

    Techniques: Western Blot

    Flow cytometry and WB analysis targeting the exosome marker CD9 on isolated vesicles. CD9 positive vesicles were detected by flow cytometry. Median fluorescence intensity (MFI) was reported as a signal to noise (S/N) ratio to isotype control in EVs isolated from E10 (A), BxPC3 (B) and H3 (C) cells (n = 3). The presence of CD9 was also analyzed by WB, which was detected in vesicles from E10 (D) and BxPC3 cells (E) (n = 3).

    Journal: PLoS ONE

    Article Title: Efficient extracellular vesicle isolation by combining cell media modifications, ultrafiltration, and size-exclusion chromatography

    doi: 10.1371/journal.pone.0204276

    Figure Lengend Snippet: Flow cytometry and WB analysis targeting the exosome marker CD9 on isolated vesicles. CD9 positive vesicles were detected by flow cytometry. Median fluorescence intensity (MFI) was reported as a signal to noise (S/N) ratio to isotype control in EVs isolated from E10 (A), BxPC3 (B) and H3 (C) cells (n = 3). The presence of CD9 was also analyzed by WB, which was detected in vesicles from E10 (D) and BxPC3 cells (E) (n = 3).

    Article Snippet: Then, membranes were blocked for 1 hr at room temperature in 5% milk in TBST (Tris-buffered saline, 0.1% Tween 20) and incubated with mouse anti-CD9 antibody (10626D, Invitrogen, Carlsbad, USA) in a dilution 1:1000, overnight at 4°C.

    Techniques: Flow Cytometry, Cytometry, Western Blot, Marker, Isolation, Fluorescence

    Exosomes from patients with MS can selectively decrease the frequency of Treg cells a Representative size distribution of purified exosomes. Using a NanoSight LM10 nanoparticle analysis system, the size was analysed three times for each sample. Red error bars indicate the standard error of the mean. b Western blot analysis for CD9, CD63 and Cytochrome c proteins. The PBMC and exosome samples were collected from HC. Each lane was loaded with 3 μg of protein for blotting. c Dot plot and histogram of flow cytometry data. T cells were prepared from the peripheral blood of a healthy volunteer. They were cultured with PBS as a control or with exosomes derived from HC (HC-exosome) or patients with MS (MS-exosome) under stimulation with anti-CD3 and anti-CD28 mAbs for 48 h. IFN-γ − IL-17A − CD4 + T cells were defined as shown in the left panel, and then the expression of Foxp3 was evaluated as shown in the right panel. d The frequencies of inflammatory and regulatory T cells among CD4 + T cells after the culture described above. Among Foxp3 + CD4 + T cells, IFN-γ − IL-17A − Foxp3 + CD4 + , whereas Foxp3 + CD4 + . Data are representative of two independent experiments. A one-way ANOVA with Bonferroni’s comparison test was used for statistical analysis. Error bars represent the mean ± s.d. * p

    Journal: Nature Communications

    Article Title: Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis

    doi: 10.1038/s41467-017-02406-2

    Figure Lengend Snippet: Exosomes from patients with MS can selectively decrease the frequency of Treg cells a Representative size distribution of purified exosomes. Using a NanoSight LM10 nanoparticle analysis system, the size was analysed three times for each sample. Red error bars indicate the standard error of the mean. b Western blot analysis for CD9, CD63 and Cytochrome c proteins. The PBMC and exosome samples were collected from HC. Each lane was loaded with 3 μg of protein for blotting. c Dot plot and histogram of flow cytometry data. T cells were prepared from the peripheral blood of a healthy volunteer. They were cultured with PBS as a control or with exosomes derived from HC (HC-exosome) or patients with MS (MS-exosome) under stimulation with anti-CD3 and anti-CD28 mAbs for 48 h. IFN-γ − IL-17A − CD4 + T cells were defined as shown in the left panel, and then the expression of Foxp3 was evaluated as shown in the right panel. d The frequencies of inflammatory and regulatory T cells among CD4 + T cells after the culture described above. Among Foxp3 + CD4 + T cells, IFN-γ − IL-17A − Foxp3 + CD4 + , whereas Foxp3 + CD4 + . Data are representative of two independent experiments. A one-way ANOVA with Bonferroni’s comparison test was used for statistical analysis. Error bars represent the mean ± s.d. * p

    Article Snippet: The membranes were blocked with 5% w/v Bovine Serum Albumin (Sigma-Aldrich, MO, USA), followed by incubation overnight with primary antibodies as follows: anti-CD9 (SN4 C3-3A2, 500×, eBiosciences, CA, USA), anti-CD63 (H5C6, 500×, BioLegend, CA, USA) and anti-Cytochrome c (D18C7, 1000×, Cell Signaling Technology, MA, USA).

    Techniques: Mass Spectrometry, Purification, Western Blot, Flow Cytometry, Cytometry, Cell Culture, Derivative Assay, Expressing

    Exosomes containing viral RNA induce NKG2D ligand expression in macrophages . (A) HepG2 cells were seeded onto a 24-well plate and cultured for 24 h. EVs were isolated from 0.5 ml of cell culture medium using a polyethylene glycol method with exosome isolation kit (see Isolation of Exosomes in Experimental Procedures ) and suspended with 150 μl of 1× SDS sample buffer. The 150 μl of whole cell extract (WCE) were prepared from cultured cells. The 10 μl of EVs and 10 μl of WCE were subjected to SDS-PAGE. CD9 and β-actin proteins were detected by western blotting with anti-CD9 antibody and anti-β-actin antibody. (B) Extracellular vesicles (EVs) released from HepG2 (left) or HuH-7 (right) transfected with pHBV were collected, and the total RNA was extracted. The RNA levels of HBV RNA and GAPDH mRNA were determined by RT-qPCR and normalized against that of U6 RNA. Data are presented as mean ± SD. (C) Exosomes were isolated from EVs released from HepG2 cells using anti-CD81 antibody beads. WCE, EVs, and 10× concentrated exosomes were subjected to SDS-PAGE, and the proteins were detected by western blotting. (D) The CD81 + exosomes were isolated from EVs with anti-CD81 microbeads. The RNA levels were determined as described in (B) . Data are presented as mean ± SD ( n = 3). (E) Hepatic F4/80 + cells and hepatic NK cells were co-cultured with normal HepG2 cells or HepG2-T23 cells (HepG2-HBV), which stably express HBV, for 1 day. IFN-γ levels in the culture supernatants were determined using ELISA ( n > 3). (F) EVs released from HuH-7 or HepG2 with or without HBV were added to PMA-treated THP-1 cells (THP-1 macrophages) for 24 h. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR and normalized to GAPDH ( n = 3). (G) HepG2 cells in six-well plates were transfected with mock or pHBV and were cultured for 24 h. EVs were isolated from 5 ml of culture medium and were suspended with 100 μl of PBS. The 5 μl of EVs were mixed with 5 μl of 2× SDS sample buffer and were subjected to SDS-PAGE. The proteins were detected by western blotting with anti-CD63 antibody. (H) EVs released from HepG2 with or without HBV were added to mouse hepatic F4/80 + cells for 24 h. The expression of mRNA in hepatic F4/80 + cells was determined by RT-qPCR and normalized to GAPDH ( n = 3). (I) HepG2-NTCP cells were infected with HBV for 9 days and were subsequently co-cultured with THP-1 (transwell co-culture) for 3 days. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR.

    Journal: Frontiers in Immunology

    Article Title: Extracellular Vesicles Including Exosomes Regulate Innate Immune Responses to Hepatitis B Virus Infection

    doi: 10.3389/fimmu.2016.00335

    Figure Lengend Snippet: Exosomes containing viral RNA induce NKG2D ligand expression in macrophages . (A) HepG2 cells were seeded onto a 24-well plate and cultured for 24 h. EVs were isolated from 0.5 ml of cell culture medium using a polyethylene glycol method with exosome isolation kit (see Isolation of Exosomes in Experimental Procedures ) and suspended with 150 μl of 1× SDS sample buffer. The 150 μl of whole cell extract (WCE) were prepared from cultured cells. The 10 μl of EVs and 10 μl of WCE were subjected to SDS-PAGE. CD9 and β-actin proteins were detected by western blotting with anti-CD9 antibody and anti-β-actin antibody. (B) Extracellular vesicles (EVs) released from HepG2 (left) or HuH-7 (right) transfected with pHBV were collected, and the total RNA was extracted. The RNA levels of HBV RNA and GAPDH mRNA were determined by RT-qPCR and normalized against that of U6 RNA. Data are presented as mean ± SD. (C) Exosomes were isolated from EVs released from HepG2 cells using anti-CD81 antibody beads. WCE, EVs, and 10× concentrated exosomes were subjected to SDS-PAGE, and the proteins were detected by western blotting. (D) The CD81 + exosomes were isolated from EVs with anti-CD81 microbeads. The RNA levels were determined as described in (B) . Data are presented as mean ± SD ( n = 3). (E) Hepatic F4/80 + cells and hepatic NK cells were co-cultured with normal HepG2 cells or HepG2-T23 cells (HepG2-HBV), which stably express HBV, for 1 day. IFN-γ levels in the culture supernatants were determined using ELISA ( n > 3). (F) EVs released from HuH-7 or HepG2 with or without HBV were added to PMA-treated THP-1 cells (THP-1 macrophages) for 24 h. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR and normalized to GAPDH ( n = 3). (G) HepG2 cells in six-well plates were transfected with mock or pHBV and were cultured for 24 h. EVs were isolated from 5 ml of culture medium and were suspended with 100 μl of PBS. The 5 μl of EVs were mixed with 5 μl of 2× SDS sample buffer and were subjected to SDS-PAGE. The proteins were detected by western blotting with anti-CD63 antibody. (H) EVs released from HepG2 with or without HBV were added to mouse hepatic F4/80 + cells for 24 h. The expression of mRNA in hepatic F4/80 + cells was determined by RT-qPCR and normalized to GAPDH ( n = 3). (I) HepG2-NTCP cells were infected with HBV for 9 days and were subsequently co-cultured with THP-1 (transwell co-culture) for 3 days. The expression of mRNA in THP-1 macrophages was determined by RT-qPCR.

    Article Snippet: ExoAB antibody kit, which includes anti-CD9, anti-CD63, and anti-CD81 antibodies, was purchased from System Biosciences.

    Techniques: Expressing, Cell Culture, Isolation, SDS Page, Western Blot, Transfection, Quantitative RT-PCR, Stable Transfection, Enzyme-linked Immunosorbent Assay, Infection, Co-Culture Assay

    Membrane CD163 is associated with exosomes. ( A ) Beads-based FACS analysis of exosome-FITC and CD9-PE staining on αCD163 (dark grey) or Isotype IgG immuno-precipitated EV’s isolated from human plasma. ( B ) Negative-stain EM of exosomes purified by αCD163 or αCD9 immunoprecipitation of EV’s isolated from human plasma.

    Journal: Scientific Reports

    Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

    doi: 10.1038/srep40286

    Figure Lengend Snippet: Membrane CD163 is associated with exosomes. ( A ) Beads-based FACS analysis of exosome-FITC and CD9-PE staining on αCD163 (dark grey) or Isotype IgG immuno-precipitated EV’s isolated from human plasma. ( B ) Negative-stain EM of exosomes purified by αCD163 or αCD9 immunoprecipitation of EV’s isolated from human plasma.

    Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

    Techniques: FACS, Staining, Isolation, Purification, Immunoprecipitation

    EV-associated CD163 in mice. ( A ) CD9 and CD163 Western blot analysis of mouse plasma before (total serum) and after (non-EV fraction) precipitation of EVs (EV-fraction). Blots are cropped for presentation. Full-length blots are available in Supplementary Fig. 3 . ( B ) Beads-based FACS analysis of exosome-FITC and CD9 or CD163 co-staining on αCD163/αCD9 (dark grey) or isotype IgG immuno-precipitated EVs isolated from mouse plasma. ( C ) Levels of ectodomain sCD163 and EV-CD163 measured by mouse CD163 ELISA in Tx-114 phase-separated plasma of mice (n = 6) in models of endotoxemia, sterile thioglycollate-induced peritonitis and Listeria Monocytogenes bacteraemia.

    Journal: Scientific Reports

    Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

    doi: 10.1038/srep40286

    Figure Lengend Snippet: EV-associated CD163 in mice. ( A ) CD9 and CD163 Western blot analysis of mouse plasma before (total serum) and after (non-EV fraction) precipitation of EVs (EV-fraction). Blots are cropped for presentation. Full-length blots are available in Supplementary Fig. 3 . ( B ) Beads-based FACS analysis of exosome-FITC and CD9 or CD163 co-staining on αCD163/αCD9 (dark grey) or isotype IgG immuno-precipitated EVs isolated from mouse plasma. ( C ) Levels of ectodomain sCD163 and EV-CD163 measured by mouse CD163 ELISA in Tx-114 phase-separated plasma of mice (n = 6) in models of endotoxemia, sterile thioglycollate-induced peritonitis and Listeria Monocytogenes bacteraemia.

    Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

    Techniques: Mouse Assay, Western Blot, FACS, Staining, Isolation, Enzyme-linked Immunosorbent Assay

    CD163 is expression on EVs in human plasma. ( A ) Western blot analysis of CD9 and CD163. Blots are cropped for presentation. Full-length blots are available in Supplementary Fig. 3 . ( B ) CD163 ELISA of human plasma before (total plasma/soluble CD163) and after (Non-EV fraction/soluble ectodomain CD163) precipitation of EVs (EV fraction/EV CD163). Data presented as median with interquartile range.

    Journal: Scientific Reports

    Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

    doi: 10.1038/srep40286

    Figure Lengend Snippet: CD163 is expression on EVs in human plasma. ( A ) Western blot analysis of CD9 and CD163. Blots are cropped for presentation. Full-length blots are available in Supplementary Fig. 3 . ( B ) CD163 ELISA of human plasma before (total plasma/soluble CD163) and after (Non-EV fraction/soluble ectodomain CD163) precipitation of EVs (EV fraction/EV CD163). Data presented as median with interquartile range.

    Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

    Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    Isolation of exosomes secreted from gastric cancer cells SGC-7901 and BGC-823 treated by irradiation. A : Electron microscopic images of exosomes released from SGC-7901 and BGC-823 cell lines. Exosomes were isolated 24 h after irradiation at either 0 Gy or 6 Gy. Bars = 0.2μm. B : Western blot analysis of CD9 protein in cell lysates prepared from exosomes of SGC-7901 or BGC-823 cell line. Whole cell extract from SGC-7901 or BGC823 cells was used as negative control (NC) and GAPDH was detected as loading control.

    Journal: Journal of Cancer

    Article Title: VEGFR-2 Inhibitor Apatinib Hinders Endothelial Cells Progression Triggered by Irradiated Gastric Cancer Cells-derived Exosomes

    doi: 10.7150/jca.25370

    Figure Lengend Snippet: Isolation of exosomes secreted from gastric cancer cells SGC-7901 and BGC-823 treated by irradiation. A : Electron microscopic images of exosomes released from SGC-7901 and BGC-823 cell lines. Exosomes were isolated 24 h after irradiation at either 0 Gy or 6 Gy. Bars = 0.2μm. B : Western blot analysis of CD9 protein in cell lysates prepared from exosomes of SGC-7901 or BGC-823 cell line. Whole cell extract from SGC-7901 or BGC823 cells was used as negative control (NC) and GAPDH was detected as loading control.

    Article Snippet: After the membranes were blocked with 5% fat-free milk in TBST buffer (0.1% Tween-20) for 30 minutes at room temperature, they were incubated with antibodies against CD9 (1:1000, Abcam, USA) and GAPDH (1:1000, Abcam, USA) at 4℃ overnight.

    Techniques: Isolation, Irradiation, Western Blot, Negative Control

    Direct identification of exosomes in CCS. (a) Detection of CD9 and CD41b in CCS and CCS filtrate by western blot. Cell lysates were used as a positive control. The proteins were concentrated by trichloroacetic acid (TCA) from CCS/CCS filtrate. (b) SPRi response signals of anti-CD9/CD41b and anti-mouse IgG to cell culture medium (DMEM), CCS, and CCS filtrate. The results are representative of three independent experiments. * p

    Journal: Analytical Chemistry

    Article Title: Label-Free Quantitative Detection of Tumor-Derived Exosomes through Surface Plasmon Resonance Imaging

    doi: 10.1021/ac5023056

    Figure Lengend Snippet: Direct identification of exosomes in CCS. (a) Detection of CD9 and CD41b in CCS and CCS filtrate by western blot. Cell lysates were used as a positive control. The proteins were concentrated by trichloroacetic acid (TCA) from CCS/CCS filtrate. (b) SPRi response signals of anti-CD9/CD41b and anti-mouse IgG to cell culture medium (DMEM), CCS, and CCS filtrate. The results are representative of three independent experiments. * p

    Article Snippet: Anti-extracellular part: mouse anti-human CD9 (MAB1880, R & D), rabbit anti-human CD63 (MAB5048, R & D), mouse anti-human CD41b (555468, BD), mouse anti-human CD81 (MAB4165, R & D), mouse anti-human CD82 (MAB4616, R & D), mouse anti-human E-cadherin (AB8993, Abcam), and mouse anti-human EpCAM (MAB9601, R & D).

    Techniques: Western Blot, Positive Control, Cell Culture

    Regulation of exosome secresion. (a) Exosome secresion was suppressed by siRNA-Rab27a. SPRi response signals from anti-CD9 and anti-CD41b decreased with siRNA-Rab27a, using siRNA-NC as a negative control. Lipofectamine 2000 was used for the transfection. (b) Exosome secresion was increased by monensin treatment. The cells were treated with monensin at a concentration of 1 μM and incubated for 48 h before being detected by SPRi. Methanol, which was used to dilute monensin, was used as a negative control. SPRi response signals from anti-CD9 and anti-CD41b increased with monensin treatment. The results are representative of three independent experiments. * p

    Journal: Analytical Chemistry

    Article Title: Label-Free Quantitative Detection of Tumor-Derived Exosomes through Surface Plasmon Resonance Imaging

    doi: 10.1021/ac5023056

    Figure Lengend Snippet: Regulation of exosome secresion. (a) Exosome secresion was suppressed by siRNA-Rab27a. SPRi response signals from anti-CD9 and anti-CD41b decreased with siRNA-Rab27a, using siRNA-NC as a negative control. Lipofectamine 2000 was used for the transfection. (b) Exosome secresion was increased by monensin treatment. The cells were treated with monensin at a concentration of 1 μM and incubated for 48 h before being detected by SPRi. Methanol, which was used to dilute monensin, was used as a negative control. SPRi response signals from anti-CD9 and anti-CD41b increased with monensin treatment. The results are representative of three independent experiments. * p

    Article Snippet: Anti-extracellular part: mouse anti-human CD9 (MAB1880, R & D), rabbit anti-human CD63 (MAB5048, R & D), mouse anti-human CD41b (555468, BD), mouse anti-human CD81 (MAB4165, R & D), mouse anti-human CD82 (MAB4616, R & D), mouse anti-human E-cadherin (AB8993, Abcam), and mouse anti-human EpCAM (MAB9601, R & D).

    Techniques: Negative Control, Transfection, Concentration Assay, Incubation

    Correlation between exosome secretion and metastatic potential. (a) SPRi response signals from anti-CD9 and anti-CD41b were higher in CCS from MHCC97H than that from MHCC97L. (b) Western blot detection of CD9 and CD41b in exosomes secreted from MHCC97H and MHCC97L cells. Exosomes were isolated from the same number (5 × 10 7 ) of MHCC97H and MHCC97L cells. The purified exosomes were resuspended in lysis buffer and analyzed by western blot. (c) Quantification of the relative protein levels from the gray scan of the western blot in panel b. The expression level of each protein in MHCC97H was treated as 100, and the relative expression level in MHCC97L cells was calculated accordingly. (d) SPRi response signals of anti-CD9/CD41b/MET were higher in CCS from B16-F10 than in that from B16-F1. All histograms are representative of three independent experiments. * p

    Journal: Analytical Chemistry

    Article Title: Label-Free Quantitative Detection of Tumor-Derived Exosomes through Surface Plasmon Resonance Imaging

    doi: 10.1021/ac5023056

    Figure Lengend Snippet: Correlation between exosome secretion and metastatic potential. (a) SPRi response signals from anti-CD9 and anti-CD41b were higher in CCS from MHCC97H than that from MHCC97L. (b) Western blot detection of CD9 and CD41b in exosomes secreted from MHCC97H and MHCC97L cells. Exosomes were isolated from the same number (5 × 10 7 ) of MHCC97H and MHCC97L cells. The purified exosomes were resuspended in lysis buffer and analyzed by western blot. (c) Quantification of the relative protein levels from the gray scan of the western blot in panel b. The expression level of each protein in MHCC97H was treated as 100, and the relative expression level in MHCC97L cells was calculated accordingly. (d) SPRi response signals of anti-CD9/CD41b/MET were higher in CCS from B16-F10 than in that from B16-F1. All histograms are representative of three independent experiments. * p

    Article Snippet: Anti-extracellular part: mouse anti-human CD9 (MAB1880, R & D), rabbit anti-human CD63 (MAB5048, R & D), mouse anti-human CD41b (555468, BD), mouse anti-human CD81 (MAB4165, R & D), mouse anti-human CD82 (MAB4616, R & D), mouse anti-human E-cadherin (AB8993, Abcam), and mouse anti-human EpCAM (MAB9601, R & D).

    Techniques: Western Blot, Isolation, Purification, Lysis, Expressing

    Characterization and identification of exosomes purified from MHCC97H cell CCS. (a) Transmission electron microscopy (TEM) characterization of purified exosomes. The mean diameter of exosomes was ∼70 nm. (b) Detection of purified exosomes through antibody microarray sensor chip. SPRi sensorgrams showing binding of exosomes to various antibodies: anti-CD9, CD41b, CD63, CD82, E-cadherin, EpCAM, and anti-mouse IgG. Reflective index (μRIU) changes are plotted as a function of time (s). Anti-mouse IgG was used as a negative control. (c) Western blot detection of the expression of CD9 and CD41b in exosomes. MHCC97H cell lysates were used as a positive control.

    Journal: Analytical Chemistry

    Article Title: Label-Free Quantitative Detection of Tumor-Derived Exosomes through Surface Plasmon Resonance Imaging

    doi: 10.1021/ac5023056

    Figure Lengend Snippet: Characterization and identification of exosomes purified from MHCC97H cell CCS. (a) Transmission electron microscopy (TEM) characterization of purified exosomes. The mean diameter of exosomes was ∼70 nm. (b) Detection of purified exosomes through antibody microarray sensor chip. SPRi sensorgrams showing binding of exosomes to various antibodies: anti-CD9, CD41b, CD63, CD82, E-cadherin, EpCAM, and anti-mouse IgG. Reflective index (μRIU) changes are plotted as a function of time (s). Anti-mouse IgG was used as a negative control. (c) Western blot detection of the expression of CD9 and CD41b in exosomes. MHCC97H cell lysates were used as a positive control.

    Article Snippet: Anti-extracellular part: mouse anti-human CD9 (MAB1880, R & D), rabbit anti-human CD63 (MAB5048, R & D), mouse anti-human CD41b (555468, BD), mouse anti-human CD81 (MAB4165, R & D), mouse anti-human CD82 (MAB4616, R & D), mouse anti-human E-cadherin (AB8993, Abcam), and mouse anti-human EpCAM (MAB9601, R & D).

    Techniques: Purification, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Microarray, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Western Blot, Expressing, Positive Control

    Optimization of tetraspanin antibody combination. 4∙10 9 particles of PC3-derived EVs were captured onto either anti-CD9 (Clone VJ1/20) or anti-CD63 (Clone TEA3/18) coated beads followed by detection with PE-conjugated antibodies directed against CD9, CD81 or CD63. The sensitivity of each antibody combination is compared using the Stain Index (SI) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$SI=\frac{MFI\,positive\mbox{--}MFI\,background}{2\sigma \,background}$$\end{document} S I = M F I p o s i t i v e – M F I b a c k g r o u n d 2 σ b a c k g r o u n d , where σ is the standard deviation and MFI Mean Fluorescence Intensity; and the Relative fluorescence Index (RFI), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RFI=\frac{MFI\,positive}{MFI\,background}$$\end{document} R F I = M F I p o s i t i v e M F I b a c k g r o u n d , indicated on the upper right corner of each panel. A representative experiment out of 3 is shown.

    Journal: Scientific Reports

    Article Title: High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

    doi: 10.1038/s41598-019-38516-8

    Figure Lengend Snippet: Optimization of tetraspanin antibody combination. 4∙10 9 particles of PC3-derived EVs were captured onto either anti-CD9 (Clone VJ1/20) or anti-CD63 (Clone TEA3/18) coated beads followed by detection with PE-conjugated antibodies directed against CD9, CD81 or CD63. The sensitivity of each antibody combination is compared using the Stain Index (SI) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$SI=\frac{MFI\,positive\mbox{--}MFI\,background}{2\sigma \,background}$$\end{document} S I = M F I p o s i t i v e – M F I b a c k g r o u n d 2 σ b a c k g r o u n d , where σ is the standard deviation and MFI Mean Fluorescence Intensity; and the Relative fluorescence Index (RFI), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$RFI=\frac{MFI\,positive}{MFI\,background}$$\end{document} R F I = M F I p o s i t i v e M F I b a c k g r o u n d , indicated on the upper right corner of each panel. A representative experiment out of 3 is shown.

    Article Snippet: After the binding step, beads were stained with either anti-CD63 (Clone TEA3/18), anti-CD81 (MEM-38) or anti-CD9 (Clone VJ1/20) antibodies (Immunostep, S.L.), either biotinylated or PE-conjugated.

    Techniques: Derivative Assay, Staining, Standard Deviation, Fluorescence

    Direct detection of EpCAM in urinary EVs. ( A ) EpCAM detection on PC3-derived EVs. 10–20 µg (4.59–9.18∙10 9 particles) of PC3-derived EVs were captured onto anti-CD63-coated beads followed by detection with biotinylated anti-EpCAM antibody by flow cytometry. ( B ) 5–10 µg (2.29–4.59∙10 9 particles) of PC3-derived EVs were captured onto anti-EpCAM-coated beads followed by detection with biotinylated anti-CD9 antibody by flow cytometry. ( C ) Specificity of anti-EpCAM-coated beads: antibody blocking. To confirm the specificity of anti-EpCAM-coated beads, PC3-derived EVs were pre-incubated with anti-EpCAM antibody, previously to their incubation with microbeads. ( D ) Specificity of anti-EpCAM-coated beads: negative control. 1.78∙10 9 particles of SK-Mel-28-derived EVs (EpCAM-negative cell line) were captured onto either anti-CD63-coated beads, anti-EpCAM- or IgG1-coated beads followed by detection with biotinylated anti-CD81 antibody by flow cytometry. ( E ) Detection of EpCAM by WB in EVs from 8 ml of urine. 8 ml of pre-treated urine from 3 healthy donors (HD) and 6 patients (P1- P6) were used to purify EVs by ultra-centrifugation and they were analysed by WB. The number under the EpCAM panel corresponds to the Relative fluoresce Index (RFI) in the flow cytometry experiment. ( F ) Detection of EpCAM by flow cytometry in EVs from 500 µl of urine. 500 µl of pre-treated urine were incubated with either IgG1 or anti- EpCAM-coated beads followed by detection with biotinylated anti-CD9 antibody and flow cytometry analysis. Three flow cytometry experiments were performed using the same patient samples and the results from a representative experiment are shown.

    Journal: Scientific Reports

    Article Title: High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

    doi: 10.1038/s41598-019-38516-8

    Figure Lengend Snippet: Direct detection of EpCAM in urinary EVs. ( A ) EpCAM detection on PC3-derived EVs. 10–20 µg (4.59–9.18∙10 9 particles) of PC3-derived EVs were captured onto anti-CD63-coated beads followed by detection with biotinylated anti-EpCAM antibody by flow cytometry. ( B ) 5–10 µg (2.29–4.59∙10 9 particles) of PC3-derived EVs were captured onto anti-EpCAM-coated beads followed by detection with biotinylated anti-CD9 antibody by flow cytometry. ( C ) Specificity of anti-EpCAM-coated beads: antibody blocking. To confirm the specificity of anti-EpCAM-coated beads, PC3-derived EVs were pre-incubated with anti-EpCAM antibody, previously to their incubation with microbeads. ( D ) Specificity of anti-EpCAM-coated beads: negative control. 1.78∙10 9 particles of SK-Mel-28-derived EVs (EpCAM-negative cell line) were captured onto either anti-CD63-coated beads, anti-EpCAM- or IgG1-coated beads followed by detection with biotinylated anti-CD81 antibody by flow cytometry. ( E ) Detection of EpCAM by WB in EVs from 8 ml of urine. 8 ml of pre-treated urine from 3 healthy donors (HD) and 6 patients (P1- P6) were used to purify EVs by ultra-centrifugation and they were analysed by WB. The number under the EpCAM panel corresponds to the Relative fluoresce Index (RFI) in the flow cytometry experiment. ( F ) Detection of EpCAM by flow cytometry in EVs from 500 µl of urine. 500 µl of pre-treated urine were incubated with either IgG1 or anti- EpCAM-coated beads followed by detection with biotinylated anti-CD9 antibody and flow cytometry analysis. Three flow cytometry experiments were performed using the same patient samples and the results from a representative experiment are shown.

    Article Snippet: After the binding step, beads were stained with either anti-CD63 (Clone TEA3/18), anti-CD81 (MEM-38) or anti-CD9 (Clone VJ1/20) antibodies (Immunostep, S.L.), either biotinylated or PE-conjugated.

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Blocking Assay, Incubation, Negative Control, Western Blot, Centrifugation

    Specificity of EV immunocapture on antibody-coated microbeads. ( A ) Gating strategy. EVs immobilised on 6 μm APC-beads were stained using biotinylated antibody followed by PE-conjugated streptavidin and analysed by flow cytometry. A gate containing only single beads was created in the Forward Scatter (FSC)/Side Scatter (SSC) plot. A second gate, within single beads, confirmed the APC fluorescence of microbeads. 1500–2000 events from this combined gating were acquired and analysed for PE labelling. ( B ) Negative control, IgG1. 10 9 particles of PC3-derived EVs were captured onto either anti-CD63 (Clone TEA3/18) or IgG1-coated beads followed by detection with biotinylated antibody directed against CD9. A sample with no EVs is also shown for comparison. ( C ) Antibody blocking. 10 9 particles of PC3-derived EVs were pre-incubated with increasing amounts of the indicated soluble blocking antibody [anti-CD63 (Clone TEA3/18), anti-CD9 (Clone VJ1/20)] before being incubated for capture on CD63- (left) or CD9- (right) coated beads. Experiments are representative of 3 independent repetitions.

    Journal: Scientific Reports

    Article Title: High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

    doi: 10.1038/s41598-019-38516-8

    Figure Lengend Snippet: Specificity of EV immunocapture on antibody-coated microbeads. ( A ) Gating strategy. EVs immobilised on 6 μm APC-beads were stained using biotinylated antibody followed by PE-conjugated streptavidin and analysed by flow cytometry. A gate containing only single beads was created in the Forward Scatter (FSC)/Side Scatter (SSC) plot. A second gate, within single beads, confirmed the APC fluorescence of microbeads. 1500–2000 events from this combined gating were acquired and analysed for PE labelling. ( B ) Negative control, IgG1. 10 9 particles of PC3-derived EVs were captured onto either anti-CD63 (Clone TEA3/18) or IgG1-coated beads followed by detection with biotinylated antibody directed against CD9. A sample with no EVs is also shown for comparison. ( C ) Antibody blocking. 10 9 particles of PC3-derived EVs were pre-incubated with increasing amounts of the indicated soluble blocking antibody [anti-CD63 (Clone TEA3/18), anti-CD9 (Clone VJ1/20)] before being incubated for capture on CD63- (left) or CD9- (right) coated beads. Experiments are representative of 3 independent repetitions.

    Article Snippet: After the binding step, beads were stained with either anti-CD63 (Clone TEA3/18), anti-CD81 (MEM-38) or anti-CD9 (Clone VJ1/20) antibodies (Immunostep, S.L.), either biotinylated or PE-conjugated.

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence, Negative Control, Derivative Assay, Blocking Assay, Incubation

    Dynamic range and limit of detection. EVs were captured on anti-CD63-coated beads followed by detection with biotinylated anti-CD9 antibody (Clone VJ1/20). ( A ) Flow cytometry analysis profiles. Increasing amounts (30 ng to 10 μg; 1.37∙10 7 –4.59∙10 9 particles) of PC3-derived EVs were captured on 6 μm beads and detected by flow cytometry. The total volume of the assay was 100 μl. The graph represents the overlay of the curves obtained. The legend indicates the amount of EVs and the RFI for each curve. ( B ) Regression analysis. RFI (right) and SI values (left) were plotted as a scatter graph and fitted to a polynomic curve (upper panel). Linearity can be observed between 0.2–8 µg of PC3-derived EVs with a r 2 > 0.98 (lower panels). The minimal amount of EVs detected (RFI > 1), corresponds to 30 ng yielding a RFI of 1.08. A representative experiment out of 3 is shown.

    Journal: Scientific Reports

    Article Title: High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

    doi: 10.1038/s41598-019-38516-8

    Figure Lengend Snippet: Dynamic range and limit of detection. EVs were captured on anti-CD63-coated beads followed by detection with biotinylated anti-CD9 antibody (Clone VJ1/20). ( A ) Flow cytometry analysis profiles. Increasing amounts (30 ng to 10 μg; 1.37∙10 7 –4.59∙10 9 particles) of PC3-derived EVs were captured on 6 μm beads and detected by flow cytometry. The total volume of the assay was 100 μl. The graph represents the overlay of the curves obtained. The legend indicates the amount of EVs and the RFI for each curve. ( B ) Regression analysis. RFI (right) and SI values (left) were plotted as a scatter graph and fitted to a polynomic curve (upper panel). Linearity can be observed between 0.2–8 µg of PC3-derived EVs with a r 2 > 0.98 (lower panels). The minimal amount of EVs detected (RFI > 1), corresponds to 30 ng yielding a RFI of 1.08. A representative experiment out of 3 is shown.

    Article Snippet: After the binding step, beads were stained with either anti-CD63 (Clone TEA3/18), anti-CD81 (MEM-38) or anti-CD9 (Clone VJ1/20) antibodies (Immunostep, S.L.), either biotinylated or PE-conjugated.

    Techniques: Flow Cytometry, Cytometry, Derivative Assay

    Characterization of PC3-derived EVs. ( A ) Nanoparticle tracking analysis (NTA). Average size and concentration were obtained in a Nanosight equipment capturing 3 videos of 60 s per measurement, with a focus −15 to +15 and camera level 12. ɸ: diameter. ( B ) Western Blot. EVs were loaded on SDS-PAGE and immunoblotted for β-actin (Sigma) and antibodies against tetraspanins [anti-CD9 (MEM62), -CD63 (MEM259) and -CD81 (MEM-38)]. Three gels were loaded: one gel, under non-reducing conditions with 2.2·10 9 particles, for CD9 and CD81 detection, exposed for 2 min; a second non-reducing gel with 6.8 · 10 9 particles, for CD63 detection, exposed for 1 h; and the third gel under reducing conditions, for actin detection, exposed for 40 s. The experiment shown is representative of 4.

    Journal: Scientific Reports

    Article Title: High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

    doi: 10.1038/s41598-019-38516-8

    Figure Lengend Snippet: Characterization of PC3-derived EVs. ( A ) Nanoparticle tracking analysis (NTA). Average size and concentration were obtained in a Nanosight equipment capturing 3 videos of 60 s per measurement, with a focus −15 to +15 and camera level 12. ɸ: diameter. ( B ) Western Blot. EVs were loaded on SDS-PAGE and immunoblotted for β-actin (Sigma) and antibodies against tetraspanins [anti-CD9 (MEM62), -CD63 (MEM259) and -CD81 (MEM-38)]. Three gels were loaded: one gel, under non-reducing conditions with 2.2·10 9 particles, for CD9 and CD81 detection, exposed for 2 min; a second non-reducing gel with 6.8 · 10 9 particles, for CD63 detection, exposed for 1 h; and the third gel under reducing conditions, for actin detection, exposed for 40 s. The experiment shown is representative of 4.

    Article Snippet: After the binding step, beads were stained with either anti-CD63 (Clone TEA3/18), anti-CD81 (MEM-38) or anti-CD9 (Clone VJ1/20) antibodies (Immunostep, S.L.), either biotinylated or PE-conjugated.

    Techniques: Derivative Assay, Concentration Assay, Western Blot, SDS Page

    Detection of EVs from healthy donors urine. ( A ) Purified EVs from urine. 68 µg (6.8∙10 10 particles) of EVs from healthy donors (Hansa Biomed) were analysed by Western Blot for detection of CD63 (1 h exposure), CD81 (10 min exposure), CD9 (10 s exposure) and β-actin (10 min exposure) (left). 10 µg (10 9 particles) were captured onto anti-CD63-coated beads followed by detection with biotinylated anti-CD9 antibody by flow cytometry (right). ( B ) EVs contained in 500 µl of urine analysed by flow cytometry. 500 µl of healthy donor urine was pre-treated by mild reduction (see methods) and incubated with either anti-CD63- or IgG1-coated beads followed by detection with biotinylated anti-CD9 antibody by flow cytometry. Stain Index (SI) and the Relative fluoresce Index (RFI) are indicated inside each panel. A representative experiment out of 3 is shown.

    Journal: Scientific Reports

    Article Title: High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

    doi: 10.1038/s41598-019-38516-8

    Figure Lengend Snippet: Detection of EVs from healthy donors urine. ( A ) Purified EVs from urine. 68 µg (6.8∙10 10 particles) of EVs from healthy donors (Hansa Biomed) were analysed by Western Blot for detection of CD63 (1 h exposure), CD81 (10 min exposure), CD9 (10 s exposure) and β-actin (10 min exposure) (left). 10 µg (10 9 particles) were captured onto anti-CD63-coated beads followed by detection with biotinylated anti-CD9 antibody by flow cytometry (right). ( B ) EVs contained in 500 µl of urine analysed by flow cytometry. 500 µl of healthy donor urine was pre-treated by mild reduction (see methods) and incubated with either anti-CD63- or IgG1-coated beads followed by detection with biotinylated anti-CD9 antibody by flow cytometry. Stain Index (SI) and the Relative fluoresce Index (RFI) are indicated inside each panel. A representative experiment out of 3 is shown.

    Article Snippet: After the binding step, beads were stained with either anti-CD63 (Clone TEA3/18), anti-CD81 (MEM-38) or anti-CD9 (Clone VJ1/20) antibodies (Immunostep, S.L.), either biotinylated or PE-conjugated.

    Techniques: Purification, Western Blot, Flow Cytometry, Cytometry, Incubation, Staining