Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 1. Expression of CD9 in mouse testis. A) Characterization of anti-CD9 antibody-purified mouse testis cells using flow cytometry. Unselected (upper row) and anti-CD9 antibody-selected (lower row) testis cells were stained with FITC-conjugated streptavidin, PE-conjugated anti-a6-integrin antibody, or APC-conjugated anti-c-kit antibody, and the fluorescence was compared with that of controls. The dotted lines show the range of fluores- cence in the controls. Note the increased percentages of all three antigens in the anti-CD9 antibody-selected cell population. B) Immunohistological staining of CD9 antigen in mouse testis. Positive cells were found on the basement membrane of seminiferous tubules (arrows). Staining was also observed in the interstitial cells. Bar 5 50 mm (section). Stain: DAB followed by hematoxylin.

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Cytometry, Staining, Membrane

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 2. Expression of CD9 on mouse spermatogonial stem cells. A) Gross and histological appearance of recipient testes after transplantation of LacZ-marked donor testis cells. Left: Testis from a W recipient mouse 2 mo after transplantation of anti-CD9 antibody-selected cells. Middle: Testis from a W recipient mouse 2 mo after transplantation of unselected testis cells. Right: Histological section from a W recipient testis 2 mo after transplantation of anti-CD9-antibody-selected testis cells. Note the normal appearance and organization of spermatogenesis. Stain: X-gal followed by hematoxylin and eosin. B) Enhanced colonization of recipient testis by anti-CD9 antibody-selected mouse testis cells. The degree of coloni- zation in three experiments is indicated by the number of individual blue colonies. The values (mean 6 SEM) for anti-CD9 antibody-selected cells and unselected cells were 5.5 6 0.8 (n 5 16) and 0.8 6 0.2 (n 5 18) cells per 3 3 104 injected cells, respectively. Bars 5 1 mm (testes) and 50 mm (section).

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Transplantation Assay, Staining, Injection

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 3. Expression of CD9 on rat spermatogonial stem cells. A) Characterization of anti-CD9 antibody-purified rat testis cells using flow cytometry. Unselected (left) and anti-CD9 antibody-selected (right) testis cells were stained with Cy5-conjugated anti-mouse IgG antibody, and the fluorescence was compared with similar cell controls. The dotted lines show the control range of fluorescence. Note the increase in percentage of CD9-positive cells after magnetic selection. B) The appearance of recipient testes after transplantation of green fluorescent protein-marked donor testis cells. Seminiferous tubules were dissected using fine forceps. Left: Testis from a busulfan-treated nude recipient mouse 3 mo after transplantation of unselected testis cells (8.2 3 104 cells injected). Right: Testis from a busulfan-treated nude recipient mouse 3 mo after transplantation of anti-CD9 antibody-selected cells (2.7 3 104 cells injected). Note the increase in number of colonies of CD9-positive cells despite the lower concentration of injected cells. Bar 5 100 mm. C) Enhanced colonization of recipient testis by anti-CD9 antibody-selected rat testis cells. The number of individual fluorescent colonies represents the degree of colonization in three experiments. The values (mean 6 SEM) for anti-CD9 antibody-selected and unselected cells were 15.8 6 4.2 (n 5 16) and 3.1 6 0.9 (n 5 13) cells per 105 injected, respectively.

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Cytometry, Staining, Control, Selection, Transplantation Assay, Injection, Concentration Assay

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: EVs attach to the surface of C. albicans . (a) Human CD9-positive MEV Ca (left) or MEV (right) (each from 5 × 10 5 monocytes) adhered to the surface of C. albicans , as seen in fixed imaging (CLSM). Blue, C. albicans ; red, CD9. Bars, 10 μm. Data are representative of n = 4 independent experiments. (b) C. albicans -induced EVs bound more abundantly than EVs from untreated PBMCs to C. albicans . Stained C. albicans are shown to exclude staining effects. (c) C. albicans -induced EVs from PBMCs exposed CR1, CD9, and CD63 on the surface. (d) Inhibition of CR1 on the EVs substantially reduced the attachment of EVs to C. albicans . (e) EVs from C. albicans -induced PBMCs (PEV Ca ) are substantially reduced when C. albicans is not opsonized (-op). Data in panels b, d, and e represent mean values ± SD, unpaired two-tailed t test; n = 4 independent experiments.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Imaging, Staining, Inhibition, Two Tailed Test

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: hsa-miR-24-3p increases C. albicans growth. (a) Growth of C. albicans was measured after the fungus was incubated with MEV Ca or MEV. Growth was monitored over 30 h in a plate reader, which measured the OD every hour. Data are presented as mean values ± SD; n = 3 different donors. (b) C. albicans hyphae grew in the presence of MEV Ca but not MEV, as observed by CLSM. Blue, C. albicans . Bars, 10 μm. Data are representative of n = 4 independent experiments. (c) C. albicans hyphae, measured using the ZEN 2011 software, were significantly longer in the presence of MEV Ca than MEV. Data are presented as mean values ± SD, P < 0.0001; n = 17; from n = 3 different experiments. (d) RNA from CD9-positive vesicles also migrated into C. albicans , as observed in CLSM. (e) RNA from MEV Ca migrated into C. albicans , as observed by 3D SIM. For panels d and e, data are representative of n = 3 independent experiments. Blue, C. albicans ; green, RNA; red, CD9. Bars, 10 μm. (f) hsa-miR-24-3p together with fluorescently labeled hsa-miR-24-3p sensor migrates into C. albicans (lower) but significantly less when hsa-miR24-3p was inhibited in monocytes (upper). (g) Mean values ± SD of fluorescence from 5 independent experiments as measured by a plate reader are shown on the right ( P = 0.003, unpaired two-tailed t test). (h and i) Hyphal growth of C. albicans in the presence of hsa-miR-24-3p. Blue, C. albicans . Bars, 10 μm. Data are presented as mean values ± SD; P < 0.0001; n = 20; n = 4 different experiments. (j and k) Incubation with hsa-miR-24-3p increased growth of both C. albicans and human hepatic cancer cells (HEPG2), as determined by metabolic activity (CellTiter-Blue assay). Data are presented as mean values ± SD; P = 0.0093 and P = 0.0027; unpaired two-tailed t test; n = 3 different experiments.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Incubation, Software, Labeling, Fluorescence, Two Tailed Test, Activity Assay, CtB Assay

Journal: Molecular & Cellular Proteomics

Article Title: Proteomics Analysis of Bladder Cancer Exosomes

doi: 10.1074/mcp.m000063-mcp201

Figure Lengend Snippet: FIG. 1. Characterization of HT1376-derived exosomes using Western blotting, flow cytometry, and electron microscopy. Cell (CL) and exosome (Exo) lysates (5 g/well) were compared by Western blotting using a range of antibodies as indicated. This demonstrated relative enrichment of several proteins in exosomes. Some markers, such as gp96, were absent from exosomes, indicating negligible contamination of the preparations by cellular debris (this is representative of three experiments) (A). Exosomes coupled to latex beads were analyzed by flow cytometry, and this revealed positive expression of tetraspanin molecules on the exosome surface. Median fluorescence intensity values (MFI) are shown (representative of 5 experiments) (B). Intentional contamination of purified exosomes with increasing amounts of FBS prior to coupling to latex beads reveals a decrease in signal intensity for CD9 (mean S.E.; n 6; **, p 0.001, one-way analysis of variance with Tukey’s post test) (B, line graph). Material pelleted at 70,000 g from cell-conditioned medium was overlaid on a linear sucrose gradient (0.2–2.02 M) and ultracentrifuged for 18 h at 210,000 g. Collected fractions were analyzed by refractometry to ascertain fraction density and thereafter by Western blot using antibodies to TSG101, which is an exosome marker. TSG101 floats at typical exosome densities of between 1.1 and 1.2 g/ml (representative of four experiments) (C). Transmission electron micrograph of a typical exosome preparation reveals heterogeneous vesicles between 30 and 100 nm in diameter (D). CK, cytokeratin.

Article Snippet: The following primary monoclonal antibodies were used: TSG101, lysosomeassociated membrane protein 1 (LAMP-1), hsp90, calnexin, HLA-G, galectin-3, basigin, hnRNPK, gp96, cytokeratins 18 and 17, and CD44 (Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (BioChain Institute, Inc.), CD9 (R&D Systems), and CD63 and CD81 (Serotec).

Techniques: Derivative Assay, Western Blot, Flow Cytometry, Electron Microscopy, Expressing, Fluorescence, Purification, Marker, Transmission Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: A negative feedback loop between fibroadipogenic progenitors and muscle fibres involving endothelin promotes human muscle fibrosis

doi: 10.1002/jcsm.12974

Figure Lengend Snippet: Antibody panel used for CyTOF

Article Snippet: CD9 , 171Yb , SN4 C3‐3A2 , Fluidigm , 3171009B.

Techniques:

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Western blot showing transmembrane proteins (CD9, CD63, and CD81), a cytosolic protein (syntenin-1) and two “negative” controls (AChE and calnexin) in cell lysates (CL) and the pellets obtained from HeLa 24 h conditioned medium after differential ultracentrifugation (2 K, 10 K, 200 K). The loaded material comes from 20 × 10 6 cells for the centrifugation pellets, and from 0.2 × 10 6 cells for the cell lysate. Representative of 3 independent experiments. b NTA and EM analysis of 200 K pellets obtained from HeLa 24 h conditioned medium. The quantifications represent the mean of concentration or frequency of EVs of different diameters, error bars show the standard deviation (SD). N = 3 independent experiments. Scale bar of the zooms: 0.1 μm. c Principle of immunoprecipitation of CD63 and CD9 EVs in HeLa concentrated conditioned medium and representative Western blot of the pull-down (PD) and flow-through (FT) of the immunoprecipitation, with quantification of the relative CD63 and CD9 bands intensity of three independent experiments (mean ± SD is represented). d Immuno-EM analysis of the 200 K pellet labeled with anti-CD9 (5 nm gold particles) and anti-CD63 (10 nm gold particles) antibodies. This experiment was performed once. Scale bar of the zooms: 0.1 μm. e Confocal imaging of immunofluorescence staining of CD63 and CD9 in HeLa cells. Pink arrows show CD9 localized in intracellular compartments. Representative picture of two independent experiments. Scale bar: 5 μm.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Western Blot, Centrifugation, Concentration Assay, Standard Deviation, Immunoprecipitation, Labeling, Imaging, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Principle of the RUSH system used to follow CD63 and CD9 intracellular trafficking. SBP streptavidin binding peptide, strept streptavidin, ER endoplasmic reticulum. b Micrographs and quantifications of live imaging of HeLa cells co-transfected with the CD63-mCherry and CD9-eGFP RUSH plasmids. Biotin at 40 μM was added at T = 0. White arrows show peripheral compartments where CD63 and CD9 co-localize. Z -projection of 11 planes. Scale bar: 5 μm. Quantification upon time of three independent experiments showing the mean ± SD eGFP and mCherry fluorescence intensity in the Golgi and in large compartments, the mean ± SD number of eGFP- or mCherry-positive small compartments and the median and range of the Pearson’s co-localization coefficient between eGFP and mCherry where automatically quantified. N = 3 independent experiments. 5 fields per experiments where imaged, for a total of at least 10 individual cells to analyze per experiment. c Representative electron microscopy images of HeLa cells co-transfected with RUSH constructs of CD63-mCherry and CD9-eGFP , 1 h or 2 h after incubation with biotin, or at steady-state, labeled with anti-eGFP gold 10 nm (red arrows) and anti-mCherry gold 15 nm (blue arrows). Relative labeling index (RLI) in each compartment quantified from 7 different fields per replicate is represented as mean ( n = 2 independent biological replicates). d Confocal microscopy pictures of HeLa cells ( z -projection) co-transfected with CD63-mCherry- and CD9-eGFP- RUSH plasmids, and stained with anti-Rab7 after 1 h of incubation with biotin. Scale bar: 10 μm. Mander’s coefficients representing the % of CD9 + /CD63-, CD9-/CD63 + , and CD9 + /CD63 + intracellular compartments also positive for the Rab7 signal in each cell are shown. Results from two independent experiments are shown, each dot represents one cell (23 cells from replicate 1, and 24 cells from replicate 2) and the median is represented. Ordinary one-way ANOVA with a Tukey’s multiple comparisons test was performed to compare the different categories of intracellular compartments shown on the graph.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Binding Assay, Imaging, Transfection, Fluorescence, Electron Microscopy, Construct, Incubation, Labeling, Confocal Microscopy, Staining

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Scheme of the structure and C-terminal sequences of CD63-WT and the mutant CD63-YA. b Immunofluorescence of HeLa cells transfected with the RUSH CD63-eGFP or CD63-YA-eGFP plasmids at steady state. Scale bar 10 μm. This experiment was performed once. c Micrographs of HeLa cells co-transfected with CD63-YA-eGFP and CD63-WT-mCherry or CD9-eGFP and CD63-YA-mCherry , and median ± range of the Pearson’s co-localization coefficient over time between eGFP and mCherry. Biotin was added at T = 0. Z -projection of 11 planes. Scale bar 5 μm. CD63/CD63-YA: 3 independent experiments n = 51 cells, CD9/CD63-YA: 2 independent experiments n = 33 cells. 5 fields per experiment where imaged, for a total of at least 10 individual cells to analyze per experiment.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Mutagenesis, Immunofluorescence, Transfection

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Principle and flow cytometry analysis of anti-GFP surface staining of HeLa cells transfected with the RUSH constructs CD63-WT-eGFP , CD63-YA-eGFP , or CD9-eGFP after different incubation times with biotin followed by fixation. The ratio of the surface staining (AF647) over the total GFP signal mean fluorescence intensities is represented at different time points, time 0 subtracted, mean ± SD for 3 independent experiments. b Principle and flow cytometry analysis of anti-GFP uptake after surface staining of HeLa cells transfected with the RUSH constructs CD63-WT-eGFP , CD63-YA-eGFP , or CD9-eGFP after 2 h of incubation with biotin. The mean percentage ± SD of internalized anti-GFP-AFP647 is represented for 3 independent experiments. Ordinary one-way ANOVA, Tukey’s multiple comparisons test. Gating strategy is illustrated in Supplementary Fig. .

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Flow Cytometry, Staining, Transfection, Construct, Incubation, Fluorescence

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Western blot of the cell lysate (CL) and of the different EV pellets obtained by differential ultracentrifugation of CCM from HeLa cells transfected with the RUSH plasmids CD63-WT-eGFP or CD63-YA-mCherry (24 h release with biotin). EVs from 20 × 10 6 cells and CL from 0.2 × 10 6 cells were loaded. The intensity of the band corresponding to the mCherry fusion proteins was quantified and normalized by the intensity of the CD9 band in 3 independent experiments, the mean ± SD is represented. Two-tailed paired t test. b Representative Western blot of the pull-down (PD) and flow-through (FT) of the immunoprecipitation of EVs from HeLa cells transfected with the RUSH plasmids CD63-WT-eGFP , CD63-YA-eGFP , or CD9-eGFP , recovered 24 h after biotin addition. 60 × 10 8 total particles quantified by NTA were used for each IP. Percent of GFP + cells quantified by flow cytometry were similar in the three conditions (Supplementary Fig. ). The GFP bands intensity in the PD normalized to endogenous CD9 in the corresponding PD are represented as mean ± SD for 3 independent experiments. Ordinary one-way ANOVA, Tukey’s multiple comparisons test. c Representative Western blot of EVs (200 K pellets) from HeLa cells transfected with the CD63-WT , CD63-YA , or CD9-eGFP RUSH plasmids treated with DMSO of BafA1 100 nM during 16 h. The same number of EVs between the DMSO and the BafA1 conditions were loaded on the gel (around 100 × 10 8 particles). The fold change between DMSO and BafA1 treatment for each construct is represented as mean ± SD for 3 independent experiments. Two-tailed one sample t test to compare each condition with a theoretical mean of 1. d Proportion of cellular endogenous CD9 and CD63 released in EVs, as semi-quantified on Western blots. The signal for CD9 and CD63 in 200 K pellets released by 20 × 10 6 HeLa cells was divided by the signal for the same molecule in the total lysate of 0.2 × 10 6 cells, run on the same blot. 1 representative Western blot and quantification (mean ± SD) of 3 independent experiments. Two-tailed paired t test.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Western Blot, Transfection, Two Tailed Test, Immunoprecipitation, Flow Cytometry, Construct

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: EVs were isolated by anti-GFP immuno-isolation from either non-transfected HeLa cells, or HeLa transfected with CD63-eGFP -RUSH or CD9-eGFP -RUSH, either 3 h or 24 h after biotin addition, and their composition was analyzed by mass-spectrometry. a Volcano plots representing quantified proteins with at least 2 peptides in 2 replicates in at least one condition. Shown are the fold changes of peptide abundancy between CD63- and CD9-eGFP expressing EV samples and the p -value of this quantification, for EVs recovered 3 h (left) or 24 h (right) after biotin addition. Position of the membrane-associated proteins selected for further analysis is indicated. b Results of the FunRich gene enrichment analysis among the proteins either enriched in the CD63- (blue), or CD9-eGFP (red) samples, or common between the CD63 and CD9-eGFP samples (purple) at 3 h or 24 h after biotin addition. For each subcellular compartment protein list, % of proteins of this category in the list of CD63-, CD9-, or common CD63/CD9 proteins is indicated, and the p -value of this percentage being different to its counterpart in the whole HeLa cell database is calculated. P -value (hypergeometric uncorrected). c Schematic representation of the transmembrane proteins identified in the CD63-, CD9-, or CD63/CD9-eGFP EVs at 3 h and 24 h.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Isolation, Transfection, Mass Spectrometry, Expressing, Membrane

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: Names of proteins identified in EVs and used for the endosome, lysosome, and PM (plasma membrane) categories are listed.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: a Western blot showing CD9, CD63, and CD81, and the new markers LAMP1, BSG, and SLC3A2 in cell lysates (CL) and the pellets obtained from HeLa conditioned media after differential ultracentrifugation (2 K, 10 K, and 200 K). The loaded material comes from 20 × 10 6 cells for the centrifugation pellets, and from 0.2 × 10 6 cells for the cell lysate. One representative image. For each marker, mean ± SD of the quantification of the signal in 200 K pellets divided by the signal in the total lysate, run on the same blot, is shown for 3 independent experiments. Ordinary one-way ANOVA, Tukey’s multiple comparisons test. b Viability of HeLa cells at the end of the 16 h medium conditioning period in the presence of DMSO (control) or BafA1 (100 nM) or GW4869 (10 μM) drugs, measured by trypan blue in 6 independent experiments, mean ± SD is represented. No significant difference observed with an ordinary one-way ANOVA, Tukey’s multiple comparisons test. c Nanoparticle tracking analysis (NTA) of EVs obtained by differential ultracentrifugation from equal numbers of HeLa cells treated with DMSO (control), BafA1 or GW4869 during 16 h. The particles concentration according to their size and the fold change of the total particle concentration between treated and control conditions are represented as mean ± SD of 5 (200 K) or 3 (10 K) independent experiments. Two-tailed one sample t test to compare each condition with a theoretical mean of 0. d TEM analysis (1 representative image) and size measurement (in 3 independent experiments) of EVs in 200 K pellets of cells exposed to DMSO, BafA1 or GW4869. Mean ± SD of the frequency distribution of CD63 and CD9 in EVs of different diameters is represented. e Representative Western blot of cell lysates from 0.2 × 10 6 HeLa cells and EVs from 20 × 10 6 HeLa cells treated with DMSO, BafA1, or GW4869, corresponding to the samples of b – d . The mean fold change ± SD between DMSO and BafA1 or GW4869 treatment of the bands intensity in the 200 K and 10 K pellets divided by the cell lysate is represented for 6 independent experiments. Two-tailed one sample t test to compare each condition with a theoretical mean of 0.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Western Blot, Centrifugation, Marker, Control, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9

doi: 10.1038/s41467-021-24384-2

Figure Lengend Snippet: Micrographs of live imaging of HeLa cells co-transfected with either CD63-mCherry or CD9-mCherry and CD81-eGFP RUSH plasmids. Biotin was added at T = 0. The median ± range of the Pearson’s co-localization coefficient between eGFP and mCherry is represented over time after biotin addition. Scale bar: 10 μm. 2 independent experiments. 5 fields per experiment where imaged, for a total of at least 10 cells to analyze per experiment.

Article Snippet: Antibodies for immunofluorescence were mouse IgG2b anti-human CD63 (clone TS63b 1/100, available upon request to E. Rubinstein: eric.rubinstein@inserm.fr) and mouse IgG1 anti-human CD9 (clone TS9 1/100) (commercially available at Diaclone or Abcam), mouse IgG1 anti-human EEA1 (BD Transduction Laboratories, clone 14/EEA1, 1/1000), mouse IgG2a anti-human RAB5 (BD Transduction Laboratories, clone 1/Rab5, 1/100), rabbit anti-human RAB7 (Cell Signaling, D95F2, 1/100), mouse IgG1 anti-human LAMP1 (Developmental Studies Hybridoma Bank, H4A3, 1/400), goat anti-mouse IgG2b Alexafluor 647 (Invitrogen, 1/300), goat anti-mouse IgG1 Alexafluor 488 (Invitrogen 1/300), goat anti-mouse IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 568 (Invitrogen, 1/200), goat anti-rabbit IgG Alexafluor 647 (Invitrogen, 1/200).

Techniques: Imaging, Transfection

Journal: Cell reports

Article Title: Single-Cell Analysis of the Muscle Stem Cell Hierarchy Identifies Heterotypic Communication Signals Involved in Skeletal Muscle Regeneration

doi: 10.1016/j.celrep.2020.02.067

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Mouse CD9 (KMC8) , Fluidigm , Cat# 3158009B.

Techniques: Recombinant, Staining, Labeling, Software

Journal: bioRxiv

Article Title: Exosomes are vehicles for the stress-regulated secretion of histones

doi: 10.1101/2024.04.08.588575

Figure Lengend Snippet: (A) WB of whole cell lysate (WCL, 10 μg) from a stable OLN-93 cell line expressing human CD63-EGFP and EVs (10, 20 μg) from 100,000 x g pellet. CD9, CD63, Tsg101, core histones (H2A, H2B, H3 and H4), Golgi marker GM130 as well as Actb are identified using specific antibodies. Approximate molecular mass is shown in kDa. (B-E) Immuno-TEM micrographs of OLN-93 EVs 100,000 x g .using primary antibodies as indicated and secondary antibodies conjugated to with 10 nm gold particles. (B) Anti-CD63. (C) Anti-histone H1 detected using protein A immuno-gold (10 nm) to detect the primary antibody. Scale bar in C = 200 nm. (D) Representative immuno-TEM micrographs of OLN-93 cell sections showing anti-CD63 distribution. Insert panel shows a magnified region containing an MVB. The column chart on the right shows quantification of the number of MVBs and ILVs. T otal number of MVBs versus CD63 + MVBs, and total number of ILVs versus CD63 + ILVs are shown as % of total. (E) OLN-93 sections showing immuno-TEM with anti-H1. Each data point in the column chart shows number of ILVs counted per MVB. Percentages in the column charts refer to % positively labelled ILVs compared to the total (given as 100%). Error bars represent the mean ±SD. (F) WB showing localization of a range of membrane, EV and organelle markers together with linker H1 and core histones in OptiPrep purified EVs 100,000 x g (300 µg) fractions from a 12%-36% (w/v) equilibrium density gradient. One 20 th of each fraction was loaded on the gel. Experiments were repeated 3 times, and one representative image is shown.

Article Snippet: The following antibodies were used: CD9-APC (Miltenyi Biotech, clone SN4, 130-128-037; final staining concentration 0.6 ng/μl), CD63-APC (Miltenyi Biotec, clone H5C6, 130-100-182; 0.6 ng/μL), CD81-APC (Beckman Coulter, clone JS64, A87789; 0.6 ng/μl), H3-AF488 (Invitrogen, clone 17H2L9, MA7-02023-A488; 2 ng/μL), and H4-FITC (Novus Biologicals; polyclonal, NB21-2044F; 2 ng/μL).

Techniques: Expressing, Marker, Membrane, Purification

Journal: bioRxiv

Article Title: Exosomes are vehicles for the stress-regulated secretion of histones

doi: 10.1101/2024.04.08.588575

Figure Lengend Snippet: (A) Dot plots showing r epresentative IFCM data obtained from HeLa cell conditioned media (CM) and media controls stained with either AF488 labelled anti-H3 or FITC labelled anti-H4 antibodies or stained with a mixture of APC labelled anti-CD9/CD63/CD81 antibodies to detect total tetraspanin positive EVs. (B) Quantification of detected gateable events positive for single stained H3, H4, or tetraspanins (n=3) in CM samples collected at different time points from both control and H 2 O 2 treated cell cultures. Samples were diluted post staining as indicated on the y -axes. (C) H3 co-expression and (D) H4 co-expression, post double staining with anti-CD9, -CD63, or -CD81 antibodies on CM from 24 h H 2 O 2 treated samples. See also Fig. S15. Statistical comparisons by two-way ANOVA. ****, p < 0.0001, ***, p < 0.001, **, < 0.005. (E) Radial plot showing relative abundance of H3 post-translational modifications (PTMs) identified in EVs OptiPrep versus nuclear histones. (F) Representative examples of H3 PTMs that are selectively enriched in the nucleus, or in EVs. Lysine (K) trimethylation (Me3) results from the covalent attachment of three Me groups to a K residue and is therefore a relatively “slow” modification. H3 is poorly modified with Me3 in EVs relative to nucleus regardless of whether this PTM benchmarks actively transcribed chromatin (H3K4me3) or silenced heterochromatin (H3K9me3). H3K9 is relatively unmodified in EVs, and H3K79Me2 is an example of a modification that is enriched in EV histones. Phosphorylation of H3S10 is selectively enriched in EVs from LPS treated cells.

Article Snippet: The following antibodies were used: CD9-APC (Miltenyi Biotech, clone SN4, 130-128-037; final staining concentration 0.6 ng/μl), CD63-APC (Miltenyi Biotec, clone H5C6, 130-100-182; 0.6 ng/μL), CD81-APC (Beckman Coulter, clone JS64, A87789; 0.6 ng/μl), H3-AF488 (Invitrogen, clone 17H2L9, MA7-02023-A488; 2 ng/μL), and H4-FITC (Novus Biologicals; polyclonal, NB21-2044F; 2 ng/μL).

Techniques: Staining, Control, Expressing, Double Staining, Residue, Modification, Phospho-proteomics