Journal: Frontiers in Endocrinology

Article Title: Plasma-derived exosomal miRNAs as potentially novel biomarkers for type 2 diabetes mellitus with abdominal obesity

doi: 10.3389/fendo.2025.1656132

Figure Lengend Snippet: Exosome identification. The exosomes were analyzed by (A) TEM and NTA assay (B) . (C, D) The protein expression of CD9, CD81, TSG101, Calnexin, and FABP4 in exosomes was detected by Western blot assay. Cell lysate (CL) was used as a positive control for calmodulin.

Article Snippet: Western blot analysis was performed to determine the expression of CD9 (Wuhan Sanying, China, 1:2000)、CD81 (Wuhan Sanying, China, 1:1000)、Tsg101 (Wuhan Sanying, China, 1:2000)、Calnexin (Wuhan Sanying, China, 1:5000)、FABP4 (Santa Cruz, America, 1:500)、β-actin (Wuhan Sanying, China, 1:4000).

Techniques: Expressing, Western Blot, Positive Control

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: The Journal of Biological Chemistry

Article Title: Cellular prion protein in human plasma–derived extracellular vesicles promotes neurite outgrowth via the NMDA receptor–LRP1 receptor system

doi: 10.1016/j.jbc.2022.101642

Figure Lengend Snippet: Comparison of methods for isolating EVs from human plasma. A , EVs were harvested from plasma by sequential UC. For some studies, UC EVs were further purified by molecular exclusion chromatography on Sepharose CL-6B (SEC) or by immunoaffinity chromatography on PS-specific antibody, covalently coupled to Sepharose (P-AC). B , representative NTA studies comparing the relative abundance of EVs of various sizes in UC, SEC, and P-AC EV preparations. C , TEM images of SEC and P-AC EV preparations. Specimens were negatively stained with uranyl acetate (the scale bar represents 500 nm for top images and 100 nm for bottom images ). D , immunoblot analysis of UC EVs shows the EV biomarkers, flotillin-1, heat shock protein-70 (HSP70), TSG101, CD9, and CD81. GM130 was not present, as anticipated. PrP C was detected in UC EVs by immunoblot analysis following immunoprecipitation (IP/IB). E , immunoblot analysis showing that SEC EVs and P-AC EVs retain PrP C . SEC EVs were probed for PrP C following IP/IB. Blots were probed for flotillin-1 to confirm the presence of EVs and as a control for EV protein load. EV, extracellular vesicle; GM130, golgi matrix protein 130; IB, immunoblot; NTA, nanoparticle tracking analysis; P-AC, phosphatidylserine affinity chromatography; PrP C , cellular prion protein; PS, phosphatidylserine; SEC, size-exclusion chromatography; TEM, transmission electron microscopy; TSG101, tumor susceptibility gene 101; UC, ultracentrifugation.

Article Snippet: The membranes were blocked with 5% nonfat dried milk and incubated with primary antibodies (1:1000 dilution) that detect flotillin-1 (BD Biosciences), heat shock protein-70 (Cell Signaling Technology), tumor susceptibility gene 101 (Abcam), CD9 (Novus Biologicals), CD81 (Novus Biologicals), golgi matrix protein 130 (BD Biosciences), α 2 M (Abcam), fibrinogen γ chain (Invitrogen), and PrP C (Abcam).

Techniques: Comparison, Clinical Proteomics, Purification, Chromatography, Staining, Western Blot, Immunoprecipitation, Control, Affinity Chromatography, Size-exclusion Chromatography, Transmission Assay, Electron Microscopy

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exosomal mRNAs for Angiogenic-Osteogenic Coupled Bone Repair.

doi: 10.1002/advs.202302622

Figure Lengend Snippet: Figure 2. Characterization of EVs and enriched RNAs in small EVs (sEVs). A) Surface markers (CD9, CD63, TSG101, and ARF6) and particle size distributions of EV subpopulations after purification and isolation by tangential flow filtration (TFF) and size exclusion chromatography (SEC). B) RNA distribution in different fractions of EVs. C) Particle size distributions and surface markers of isolated blank sEVs (b-sEVs), engineered sEVs (sEVsPBS), and therapeutic sEVs (t-sEVsBone RNAs) generated from blank hAdMSCs and hAdMSCs transfected by PBS buffer or bone-related pDNA cocktail, respectively. D) SEM, transmission electron microscopy (TEM), and cryogenic electron microscopy (cryo-EM) images of b-sEVs and t-sEVsBone RNAs with enriched RNAs. SEM and TEM images of b-sEVs and t-sEVsBone RNAs show no difference in the morphology of sEVs, while cryo-EM analysis suggests t-sEVsBone RNAs contain a higher RNA content. E) RNA quantity and distribution in 1 × 1012 b-sEVs, e-sEVsPBS, and t-sEVsBone RNAs (EV fraction 9–15). Synthetic mRNAs of BMP-2 and VEGF-A were used as standard samples (100 ng each mRNA). F) RT–qPCR analysis of BMP-2 and VEGF-A mRNAs indicates that t- sEVsBone RNAs contain more transcribed BMP-2 and VEGF-A mRNAs than e-sEVssPBS and b-sEVs. *p < 0.05 and ****p < 0.0001. G) Schematics of a single- sEV biochip for exosomal mRNA detection and representative total internal reflection fluorescence microscope (TIRFM) images for t-sEVsBone RNAs. Red dots: sEVs with VEGF-A mRNAs; green dots: sEVs with BMP-2 mRNAs; yellow dots: sEVs with both mRNAs (Scale bar: 10 μm). H) Colocalization percentage of t-sEVsBone RNAs with both VEGF-A and BMP-2 mRNAs (n = 15). *p < 0.05 and ****p < 0.0001. All data are presented as mean ± SD. Student’s t-test was used for comparison.

Article Snippet: After washing with PBS, the surface was incubated with a biotinylated capture antibody cocktail (20 μg mL−1 human CD63 Antibody (R&D Systems) and 20 μg mL−1 human CD9 Antibody (R&D Systems) (Table S8, Supporting Information) for 1 h at RT on the rocker.

Techniques: Isolation, Size-exclusion Chromatography, Generated, Transfection, Transmission Assay, Electron Microscopy, Cryo-EM Sample Prep, Quantitative RT-PCR, Microscopy, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exosomal mRNAs for Angiogenic-Osteogenic Coupled Bone Repair.

doi: 10.1002/advs.202302622

Figure Lengend Snippet: Figure 3. Enhanced therapeutic sEV (t-sEV) release regulated by mTORC1-autophagy axis. A) Cell-electron microscopy (Cell-EM) sections of TM-nanoEP stimulated hAdMSCs with or without plasmids show the changes of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) versus untreated hAdMSCs as a control. Quantification of cellular cryo-EM sections suggests TM-nanoEP induced activity of EV precursors, B,C) MVBs and ILVs, as evidenced by a twofold increase of the MVB number and an eightfold increase of the ILV number (n = 5). D) mRNA cargoes (BMP-2 and VEGF-A) were recognized by MB-FISH (molecular beacon-based fluorescence in-situ hybridization) probes (green). The late endosome/MVBs in hAdMSCs were stained by florescence-labeled anti-Rab7 (red). E) Colocalization quantification of mRNAs in late endosome/MVBs (n = 10). F) Western blot shows TM- nanoEP suppresses mTORC1 activity (phospho-S6 (pS6)) and thus activates autophagic activity (light chain 3-II (LC3-II). Increasing TM-nanoEP voltages results in greater reduction of mTORC1 activity and enhanced autophagy. G) Illustration of mTORC1-autophagy axis in regulating enhanced t-sEV release by TM-nanoEP. H) Western blot shows suppressed PS6 and enhanced LC3-II expression with significantly increased intracellular expression sEV marker proteins (CD63 and CD9) in wide-type hAdMSCs after TM-nanoEP. The mTORC1 and autophagic activities in TSC1/2−/−hAdMSCs were insensitive to TM-nanoEP with no impact on sEV release. I,J) sEV number and exosomal mRNA expression by TM-nanoEP stimulated TSC1/2+/+ and TSC1/2−/−

Article Snippet: After washing with PBS, the surface was incubated with a biotinylated capture antibody cocktail (20 μg mL−1 human CD63 Antibody (R&D Systems) and 20 μg mL−1 human CD9 Antibody (R&D Systems) (Table S8, Supporting Information) for 1 h at RT on the rocker.

Techniques: Electron Microscopy, Control, Cryo-EM Sample Prep, Activity Assay, In Situ Hybridization, Staining, Labeling, Western Blot, Expressing, Marker

Journal: bioRxiv

Article Title: Single Cell RNA Sequencing Reveals Emergent Notochord-Derived Cell Subpopulations in the Postnatal Nucleus Pulposus

doi: 10.1101/2023.05.21.541589

Figure Lengend Snippet: UMAP and violin plots showing significantly (*) higher expression of the surface markers A. Cd109 and B. Cd9 in late versus early stage NP cells. C. In situ fluorescence imaging of tdTomato+ cells (red), and corresponding immunofluorescence imaging of Cd109+ (green) and Cd9+ (white) cells in the NPs of mouse lumbar spines during postnatal growth. Cd9+ cells were localized to the NP periphery, with relative numbers increasing with postnatal age. Blue (DAPI) = cell nuclei; Midsagittal sections; 100µm. D. Flow cytometry analysis showing relative increases in the number of tdTomato/Krt19/Cd9+ positive cells with increasing postnatal age. E. Representative sections showing progressive accumulation of glycosaminoglycan-rich ECMs at the NP periphery with increasing postnatal age. Alcian blue and picrosirius red staining; mid-sagittal sections; scale = 100µm.

Article Snippet: Cells were stained sequentially with a rabbit polyclonal Krt19 antibody conjugated to Alexa Fluor 647 (1:200; catalog number NB100-687AF647; Novus Biologicals) and a rat monoclonal Cd9 antibody conjugated to Alexa Fluor 750 (1:200; catalog number NBP1-44876AF750; Novus) for 45 minutes on ice.

Techniques: Expressing, In Situ, Fluorescence, Imaging, Immunofluorescence, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Single Cell RNA Sequencing Reveals Emergent Notochord-Derived Cell Subpopulations in the Postnatal Nucleus Pulposus

doi: 10.1101/2023.05.21.541589

Figure Lengend Snippet: A. Representative histological sections of healthy and degenerate goat lumbar discs. Alcian blue and picrosirius red-staining; mid-sagittal sections; scale = 2mm. B. Semi-quantitative grade of healthy and degenerate goat discs. *p<0.05 vs healthy; N=3-4; Students t-test. C. Representative immunostaining of Cd9+ cells in the NPs of healthy and degenerate goat discs. Mid-sagittal sections; scale = 100µm (left) and 20µm (right). D. Quantification of the relative numbers of Cd9+ cells in the NPs of healthy and degenerate goat discs. *p<0.05 vs healthy; N=3-4; Students t-test.

Article Snippet: Cells were stained sequentially with a rabbit polyclonal Krt19 antibody conjugated to Alexa Fluor 647 (1:200; catalog number NB100-687AF647; Novus Biologicals) and a rat monoclonal Cd9 antibody conjugated to Alexa Fluor 750 (1:200; catalog number NBP1-44876AF750; Novus) for 45 minutes on ice.

Techniques: Staining, Immunostaining

Journal: bioRxiv

Article Title: Single Cell RNA Sequencing Reveals Emergent Notochord-Derived Cell Subpopulations in the Postnatal Nucleus Pulposus

doi: 10.1101/2023.05.21.541589

Figure Lengend Snippet: Schematic representations of A. the transition from early to late-stage NP cells, which is characterized by increased TGF-β and PI3k-Akt signaling, and expression of aggrecan, collagens II and VI, and Cd9; and B. emergent late-stage NP cells during postnatal growth with concomitant peripheral ECM deposition. Created with BioRender.com, individual license AG25DQC3X0.

Article Snippet: Cells were stained sequentially with a rabbit polyclonal Krt19 antibody conjugated to Alexa Fluor 647 (1:200; catalog number NB100-687AF647; Novus Biologicals) and a rat monoclonal Cd9 antibody conjugated to Alexa Fluor 750 (1:200; catalog number NBP1-44876AF750; Novus) for 45 minutes on ice.

Techniques: Expressing