Journal: Cells

Article Title: Comparative Profiling of Mouse and Human Microglial Small Extracellular Vesicles Reveals Conserved Core Functions with Distinct miRNA Signatures

doi: 10.3390/cells15020184

Figure Lengend Snippet: Robust expression exhibiting a comparative CD63-tdTomato-labeled vesicle load in resting microglia across species. ( A – C ) Confocal microscopy images of immortalized human microglia in resting state showing the presence of CD63-tdTomato-labeled vesicles (red). Cells were counterstained with Alexa Fluor™ 488 Phalloidin (green) to visualize microglial cell bodies and Hoechst (blue) to label nuclei. ( A , B ) Low-magnification view showing widespread intracellular distribution of CD63-tdTomato-labeled with majority exhibiting a perinuclear accumulation. Higher-magnification (40×) image of HuMG parent cells ( C ) demonstrating punctate CD63-positive vesicles localized along actin filaments and concentrated in the perinuclear region. ( D , E ) Resting mouse microglia exhibiting robust expression of CD63-tdTomato-labeled intracellular vesicles. ( D ) Phalloidin-488 staining defines cell boundaries and morphology, revealing widespread CD63-positive puncta throughout the cytoplasm. ( E ) Hoechst counterstaining confirms intracellular vesicle localization surrounding the nucleus. ( F ) Quantification of intracellular vesicle load in resting microglia of both mouse and human origin displayed significantly higher cellular load of CD63-expressing intracellular vesicles in microglia of mouse origin compared to that of human origin (** p < 0.01). Bars represent mean ± SEM; acquired from images obtained from independent biological replicates from each species. Scale bar: 15 µm ( A , B , D , E ) and 5 µm ( C ).

Article Snippet: Parent human (HMC3) and mouse (BV2) microglial cells stably expressing an sEV reporter were generated by transduction with a lentiviral vector encoding a CD63-tdTomato fusion (LentifectTM Purified Lentiviral Particles, Cat. # LP772-025, Genecopoeia, Rockville, MD, USA) and cultured to ~90% confluence.

Techniques: Expressing, Labeling, Confocal Microscopy, Staining

Journal: Cells

Article Title: Comparative Profiling of Mouse and Human Microglial Small Extracellular Vesicles Reveals Conserved Core Functions with Distinct miRNA Signatures

doi: 10.3390/cells15020184

Figure Lengend Snippet: Time-dependent uptake of CD63-tdTomato-labeled MGEVs by HuSCs. ( A – F ) Confocal images of HuSC exposed to purified CD63-tdTomato-labeled MsMGEVs (red) for 24 h ( A , B ), 48 h ( C , D ), or 72 h ( E , F ). Cells were stained with Alexa Fluor™ 488 Phalloidin (green) to mark the cell body and Hoechst to label nuclei (blue). Internalized CD63-tdTomato-positive sEVs appear as red puncta distributed throughout the cytoplasm and concentrated in perinuclear regions. The size of fluorescent puncta reflects intracellular endosomal accumulation of multiple internalized sEVs rather than aggregation of individual vesicles. Uptake of MsMGEVs showed a trend in an increase in sEVs overtime. Confocal images of HuSC treated with purified CD63-tdTomato-labeled HuMGEVs, red) for 24 h ( G , H ), 48 h ( I , J ), or 72 h ( K , L ). Like MsMGEVs, HuMGEV uptake was evident as intracellular red puncta; however, the overall accumulation pattern remained relatively stable across time points. ( M , N ) shows untreated HuSC microglia (control with no MGEV exposure) showing phalloidin-488 and Hoechst staining but no detectable CD63-tdTomato signal, confirming that red puncta in treated groups represent internalized CD63-tdTomato-labeled MGEVs. ( O ) Quantification of CD63-tdTomato fluorescence intensity in HuSC cells following 24 h, 48 h, or 72 h exposure to MsMGEVs or HuMGEVs. Both vesicle types were taken up by HuSC microglia, with MsMGEVs showing a modest trend toward increased accumulation over time. Bars represent mean ± SEM; individual points represent biological replicates. Scale Bar: 15 µm.

Article Snippet: Parent human (HMC3) and mouse (BV2) microglial cells stably expressing an sEV reporter were generated by transduction with a lentiviral vector encoding a CD63-tdTomato fusion (LentifectTM Purified Lentiviral Particles, Cat. # LP772-025, Genecopoeia, Rockville, MD, USA) and cultured to ~90% confluence.

Techniques: Labeling, Purification, Staining, Control, Fluorescence

Journal: Nature communications

Article Title: GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain.

doi: 10.1038/s41467-021-27056-3

Figure Lengend Snippet: Fig. 2 GAPDH binds to EV surface via G58 domain. a Western blot showing binding of G58 peptide to HEK293T (designated as 293T) and MSC EVs. The second domain of TARBP protein was attached to G58 peptide for detection by anti-TARBP2 antibody. The experiment was independently repeated two times. b NTA profile showing the size distribution of HEK293T EVs after binding to the G58T protein. Inset is the scatter plot representing size (mean) of EVs. Each dot is a mean of three reading frames taken at different timepoints. Data are shown as mean ± s.d (n = 9 independent biological experiment) Statistical differences were determined by unpaired two-sided Student’s t-test, (ns = non-significant). c Agarose-gel-shift assay of EVs after incubation with either G58T (G58 + dsRBD) protein or dsRBD of TARBP2 protein. siRNA alone was used as negative control to determine interaction of EVs with siRNA. A gradual decrease in the intensity of siRNA reflects entrapment of siRNA near the wells due to interaction with G58T EVs. Lack of siRNA binding to dsRBD treated EVs confirms G58 peptide mediated binding of protein to EV surface. The experiment was independently repeated three times. d–f High- resolution single EV analysis by Imaging Flow Cytometry (IFC) to determine localization of GAPDH and G58 peptide on EVs. d Represents method validation by using either non-labelled HEK293F derived EVs or neon GFP labelled HEK293:CD63-neon GFP derived EVs as biological reference material. e Detection of GAPDH on HEK293F, HEK293F/CD63-GFP and MSCs EVs, using alexa fluor 647 labelled anti-GAPDH antibody. f G58 peptide binding on EVs expressing GAPDH on their surface. EVs were incubated with alexa fluor 488 (af488) labelled G58 peptide and af647 anti-GAPDH antibody. Experiment (d–f) were independently repeated three times. FACS sequential gating/sorting strategies is provided as Supplementary Fig. 9. g Distribution of secreted GAPDH-GFP protein in the cell-culture media. Media from HEK293T cells expressing GAPDH-GFP protein were processed to isolate EVs from proteins by gel-filtration chromatography. Both EVs and protein fractions contained GAPDH-GFP protein, indicating vesicular and non-vesicular modes of GAPDH secretion. Data are shown as mean ± s.d (n = 3 independent biological experiments). Source data are provided as a Source Data file.

Article Snippet: In brief, 25 μl of EVs at a concentration of 1 × 1012 particles/ml were incubated overnight at 4 °C with 400 pmol AlexaFluor488 labelled G58 peptide and/or with AlexaFluor647-labelled rabbit anti human GAPDH antibodies (abcam, ab204480, clone EPR16884) or APC-labelled mouse anti human CD63 antibodies (Miltenyi Biotec, 130–100–182; clone H5C6) at a final concentration of 8 nM in v-bottom 96 wells (ThermoFisher Scientific).

Techniques: Western Blot, Binding Assay, Agarose Gel Electrophoresis, Shift Assay, Incubation, Negative Control, Imaging, Flow Cytometry, Biomarker Discovery, Derivative Assay, Expressing, Cell Culture, Chromatography

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence (V, vehicle alone) and presence of each drug (10 µM). The cells were lysed, and levels of CD9 and CD81 were examined by immunoblotting. Blots of results with fluvastatin (Fluv) and simvastatin (Simv) are shown. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) After testing 1,165 drugs, levels of CD9 and CD81 relative to actin were quantified by densitometry. Fold changes of the expression levels compared with vehicle alone were calculated and plotted. Drugs that increased the level of either CD9 or CD81 more than 1.5-fold compared with vehicle alone were regarded as positive. Correlation between fold changes in CD9 and CD81 levels was analyzed using Pearson’s correlation coefficient. ( C ) RAW264.7 cells were cultured in the absence (V) or presence of multiple statins (10 µM) and levels of CD9 and CD81 were examined by immunoblotting. The statins are arranged in order of decreasing lipophilicity. Ceri, cerivastatin; Simv, simvastatin; Fluv, fluvastatin; Ator, atorvastatin; Rosu, rosuvastatin; Prav, pravastatin. ( D ) RAW264.7 cells were cultured in the absence (shaded histograms) or presence (10 µM) of fluvastatin (open red histograms) and simvastatin (open blue histograms). Surface levels of CD9, CD63, CD81, and the integrin β1 subunit were analyzed by flow cytometry.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages

doi: 10.1371/journal.pone.0073706

Figure Lengend Snippet: ( A ) RAW264.7 cells were cultured for 24 h in the absence or presence of increasing concentrations of fluvastatin (Fluv) or simvastatin (Simv). The cells were lysed, and levels of CD9, CD63, and CD81 were examined by immunoblotting. Anti-actin blots show that comparable amounts of protein were loaded in each lane. ( B ) RAW264.7 cells were untreated (-) or cultured in the absence or presence of increasing concentrations of fluvastatin or simvastatin and stimulated for 24 h with 0.1 µg/ml LPS (+). Levels of CD9, CD63, and CD81 were examined by immunoblotting. Note that LPS downregulates CD9 and CD81 in the absence of statins (arrowheads). ( C ) RAW264.7 cells were cultured in the absence (-) or presence of 3 µM fluvastatin (+), and unstimulated (-) or stimulated for 24 h with 1 µg/ml LPS (+). mRNA levels of CD9 and CD81 were examined by reverse transcription PCR. GAPDH is an internal loading control. ( D ) RAW264.7 cells were cultured in the absence or presence of fluvastatin, and unstimulated or stimulated with LPS. Control (Cont) was an untreated culture. mRNA levels of CD9 and CD81 were examined by real-time PCR. Data shown are from one representative of three similar experiments. ( E ) Human monocytic THP-1 cells were treated for 4 h with 1 µg/ml phorbol 12-myristate 13-acetate, allowed to attach to a plate, and then cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting. ( F ) Mouse 3T3 fibroblasts were cultured in the absence or presence of increasing concentrations of simvastatin. Levels of CD9, CD63, and CD81 were examined by immunoblotting.

Article Snippet: The primary Abs were rat anti-mouse CD9 mAb (KMC8; BD Biosciences), hamster anti-mouse CD81 mAb (Eat2; UK-Serotec), rabbit anti-LAMP3 (CD63) polyclonal Ab (12632-1-AP; Proteintech), mouse anti-human CD9 mAb (MM2/57; Biosource), mouse anti-human CD81 mAb (JS64; Immunotech), rabbit anti-IκBα polyclonal Ab (9242; Cell Signaling Technology), rat anti-mouse integrin β2 subunit mAb (M18/2; BD Biosciences), rat anti-CD14 mAb (rmC5-3; BD Biosciences), rabbit anti-TLR4 polyclonal Ab (IMG577; Imgenex), mouse anti-flotillin-1 mAb (clone 18; BD Biosciences), and goat anti-CD45 polyclonal Ab (R&D Systems).

Techniques: Cell Culture, Western Blot, Reverse Transcription, Control, Real-time Polymerase Chain Reaction