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: bioRxiv

Article Title: Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI

doi: 10.1101/2023.08.17.551257

Figure Lengend Snippet: A, Proteomic analysis of VSMC-derived sEVs and EV. Venn diagram. N=3. B, Protein enrichment in the EV and sEV proteome. Heat Map. N=3. C, Western blot validation of sEV cargos. EV and sEV were isolated from VSMC’s conditioned media by differential ultracentrifugation and analysed by western blotting. Representative image from N=3. D, VSMC adhesion is regulated by collagen VI loaded to sEV. FN matrices were incubated with sEV and anti-collagen VI antibody (COLVI IgG) or control IgG. Cell adhesion was tracked by using ACEA’s xCELLigence Real-Time Cell Analysis. ANOVA, N=3. E, F, J, sEV promote directional VSMC invasion. VSMCs were treated with control siRNA (Scramble) or collagen VI-specific siRNA pools for 24h and were seeded to the FN-enriched Matrigel matrix in μ-Slide Chemotaxis assay and stained with Draq5. Cell tracking was conducted by OperaPhenix microscope for 12h and cell invasion parameters were quantified using Columbus. Kolmogorov-Smirnov test, *, p<0.05 I, Real-time PCR analysis of expression of CD9, CD63, CD81, COL6A3, EDIL3 and TGFBI in atherosclerotic plaque. *, p<0.05, Paired t-test, N=5.

Article Snippet: Antibodies were CD9 (SA35-08 clone, NBP2-67310, Novus Biologicals), CD63 (BD Pharmingen, 556019), CD81 (BD Pharmingen TM , 555676, B-11, SantaCruz, sc-166029 and M38 clone, NBP1-44861, Novus Biologicals), Syntenin-1 (Abcam, ab133267), Syndecan-4 (Abcam, ab24511), α-Actinin-4 (Abcam, ab108198), fibronectin (Abcam, ab2413, ab6328 [IST-9] (3D matrix staining) and F14 clone, ab45688 (clinical samples analysis)), β1 activating (12G10) antibody was previously described , 4B4 integrin inhibiting antibody (Beckman Coulter, 41116015), vinculin (Sigma, V9264), α5 integrin (P1D6, Abcam, ab78614), Myo10 (Sigma, HPA024223), gelatin-3BP/MAC-2BP (R&D systems, AF2226), EDIL3 antibody (R&D systems, MAB6046), TGFBI (Sigma, SAB2501486), IgG mouse (Sigma PP54), Anti-collagen Type VI antibody, clone 3C4 (Sigma, MAB1944), p34-Arc/ARPC2 antibody (Millipore, #07-227), Cortactin, LGALS3BP (R&D, AF2226), GAPDH (ab139416, Abcam).

Techniques: Derivative Assay, Protein Enrichment, Western Blot, Biomarker Discovery, Isolation, Incubation, Control, Cell Analysis, Chemotaxis Assay, Staining, Cell Tracking Assay, Microscopy, Real-time Polymerase Chain Reaction, Expressing

Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: HIV protein Nef expression in human microglia drives the release of distinct Nef-containing extracellular vesicles

doi: 10.20517/evcna.2025.106

Figure Lengend Snippet: h-microglia harboring an inducible, stably integrated Nef.GFP transgene provide an improved cellular model for investigating Nef driven vesiculation. (A) Fluorescence microscopy of Nef.GFP expression in h-microglia with a stably integrated Nef.GFP transgene under an inducible promoter, without (w/o DOX) or with (w DOX) 50 ng/mL DOX treatment for 48 h. Nuclei are labelled with DAPI (blue). Scale bar: 10 μm (white); (B) Nanoparticle tracking analysis of small (crude) EVs enriched by ultracentrifugation, after removal of the 10,000 × g pellet from the media of Nef.GFP (black) and GFP (gray) expressing h-microglia cultures. The distribution curve represents the frequency (particles per million cells) in relation to particle mode size (2r, nm); (C) Immunoblot analysis of small (crude) EVs enriched from the media of Nef.GFP and GFP expressing (+ DOX) or not (- DOX) h-microglia cultures, with antibodies directed against Nef, GFP, typical EV proteins (GAPDH, CD9) and EV impurity marker Calnexin; (D) Representative dot plots of small (crude) EVs enriched from Nef.GFP and GFP expressing cultures, displaying FITC fluorescence (FITC-A) in relation to side scatter (SS-A) after analysis with nano-flow cytometry. The EV sample enriched from LV-control h-microglia culture was used to gate fluorescent EVs. Nef.GFP+ or GFP+ EVs are indicated in red, while non-fluorescent particles are indicated in blue. Respective percentages of fluorescent particles (FITC+) from three independent experiments (± SD) are indicated in the graph. EV: Extracellular vesicles; CL: cell lysate; Nef.GFP: Nef green fluorescent protein; h-microglia: human microglia; DOX: doxycycline; DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; CD9: cluster of differentiation 9; FITC: fluorescein isothiocyanate; SS-A: side scatter; LV: lentiviral; Calnexin: calnexin protein; GFP: green fluorescent protein.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal antibodies against AChE (MAB303, Millipore, USA), Alix (2171, Cell Signaling Technology, USA), CD81 (NBP1-44861, Novus Biologicals; USA), Cytochrome C (556433, BD Biosciences, USA), Flotillin (610820, BD Biosciences, USA), GAPDH (G8795, Sigma-Aldrich, USA), GFP (sc-9996, Santa Cruz Biotechnology, USA), HIV-1 Nef (ab42355, UK), Hsp70 (ab5442, Abcam, UK), p24 (ab9071, Abcam, UK) and Tsg101 [4A10] (ab83, Abcam, UK); or goat polyclonal antibodies against Actin (sc-1615, Santa Cruz Biotechnology, USA), Annexin A2 (sc-1924) and HSC70 (sc-1059); or rabbit polyclonal antibodies against Calnexin (sc-11397), CD63 (sc-15363), HIV-1 gp120 (NBP1-76371, Novus Biologicals, USA) and HIV-1 Nef (2949, BEI Resources Repository, USA); and rabbit monoclonal antibodies against Albumin (ab192603, Abcam, UK) and CD9 (13403, Cell Signaling Technology, USA).

Techniques: Stable Transfection, Fluorescence, Microscopy, Expressing, Western Blot, Marker, Flow Cytometry, Control

Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: HIV protein Nef expression in human microglia drives the release of distinct Nef-containing extracellular vesicles

doi: 10.20517/evcna.2025.106

Figure Lengend Snippet: Inducible Nef.GFP expression in hmicroglia promotes the release of small Nef.GFP-positive EVs. (A and B) Crude EV samples enriched from the culture media of Nef.GFP and GFP expressing h-microglia, after removal of the 10,000 × g pellet, were further separated on 5%-40% iodixanol density gradient. The twelve collected fractions were analyzed by nano-flow cytometry (particles (blue) and Nef.GFP+ or GFP+ EVs (FITC + , red) per million cells), and nanoparticle tracking analysis [average mode size (nm; green) for fractions with at least 10 particles per frame]; (C and D) The same 12 fractions from Nef.GFP and GFP EV samples were also analyzed by immunoblotting with antibodies against GFP, typical EV proteins (Alix, CD81, and CD9), and impurity markers (Calnexin, Albumin and Cytochrome c). CL: Cell lysate; Nef.GFP: Nef green fluorescent protein; h-microglia: human microglia; GFP: green fluorescent protein; EV: extracellular vesicle; FITC: fluorescein isothiocyanate; Alix: ALG-2-interacting protein X; CD81: cluster of differentiation 81; CD9: cluster of differentiation 9; Calnexin: calnexin protein; Albumin: serum albumin; Cytochrome c: cytochrome c protein.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal antibodies against AChE (MAB303, Millipore, USA), Alix (2171, Cell Signaling Technology, USA), CD81 (NBP1-44861, Novus Biologicals; USA), Cytochrome C (556433, BD Biosciences, USA), Flotillin (610820, BD Biosciences, USA), GAPDH (G8795, Sigma-Aldrich, USA), GFP (sc-9996, Santa Cruz Biotechnology, USA), HIV-1 Nef (ab42355, UK), Hsp70 (ab5442, Abcam, UK), p24 (ab9071, Abcam, UK) and Tsg101 [4A10] (ab83, Abcam, UK); or goat polyclonal antibodies against Actin (sc-1615, Santa Cruz Biotechnology, USA), Annexin A2 (sc-1924) and HSC70 (sc-1059); or rabbit polyclonal antibodies against Calnexin (sc-11397), CD63 (sc-15363), HIV-1 gp120 (NBP1-76371, Novus Biologicals, USA) and HIV-1 Nef (2949, BEI Resources Repository, USA); and rabbit monoclonal antibodies against Albumin (ab192603, Abcam, UK) and CD9 (13403, Cell Signaling Technology, USA).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: LTH-sEV promotes LSEC capillarization in HFD mice and aggravates the progression of MASLD. ( A ) NTA analysis of LTH-sEV; ( B ) TEM images of LTH-sEV, scale bar = 100 nm; ( C ) Western blot analysis of CD9, CD63, Alix, TSG101, and calnexin protein expression in hepatocyte (HepG2) and LTH-sEV; ( D ) Schematic diagram of LTH-sEV injection into mice on HFD and NCD diet; ( E ) In vivo imaging detection of DiR-labeled LTH-sEV in mice; ( F ) SEM images and porosity of hepatic sinusoidal in NCD-fed mice treated with PBS, LTH-sEV, HFD-fed mice treated with PBS, LTH-sEV, n = 6, scale bar = 500 nm; ( G ) Immunohistochemistry images of CD31, Ang-2 in each group, scale bar = 50 μm, n = 6; ( H ) ELISA analysis of serum Ang-2 in each group, n = 6; ( I ) Western blot analysis of Ang-2 expression in each group, n = 3; ( J ) H&E staining, immunohistochemistry images of α-SMA, Sirius red staining in each group, scale bar = 50 μm, n = 6; ( K ) Serum ALT, AST expression in each group, n = 6; ( L ) qRT-PCR analysis of IL-1β, IL-6, and TNFα expression in each group, n = 6; ( M ) ELISA analysis of IL-1β, IL-6, and TNFα level in mice liver or serum, n = 6. Compared with HFD or NCD group, ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ns, no significance.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

Techniques: Western Blot, Expressing, Injection, In Vivo Imaging, Labeling, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Staining, Quantitative RT-PCR

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: OGT-enriched Hepatocyte-derived Extracellular Vesicles Promote Capillarization of Liver Sinusoidal Endothelial Cells in Metabolic Dysfunction-associated Steatotic Liver Disease

doi: 10.1016/j.jcmgh.2025.101721

Figure Lengend Snippet: OGT level in serum and serum sEV is positively associated with MASLD. ( A ) ELISA analysis of serum OGT expression in healthy individuals and patients with MASLD, n = 50; ( B ) ROC curve of serum OGT, AUC = .6832, P = .0016, Compared with Healthy group, ∗∗ P < .01; ( C ) Correlation analysis of serum OGT and ALT, r = .2154, P = .0438; ( D ) Correlation analysis of serum OGT and AST, r = .2493, P = .0171; ( E ) Correlation analysis of serum OGT and TG, r = .2541, P = .0197; ( F ) Immunohistochemical images of OGT expression in liver of healthy individuals and patients with MASLD; ( G ) Schematic diagram of the collection of Healthy-sEV and MASLD-sEV; ( H–I ) NTA and TEM detection of Healthy-sEV and MASLD-sEV, scale bar = 100 nm; ( J ) Western blot analysis of CD9, CD63, Alix and TSG101 protein expression in Healthy-sEV and MASLD-sEV; ( K ) Western blot analysis of OGT protein expression in Healthy-sEV and MASLD-sEV, n = 3; ( L ) ELISA analysis of of OGT in Healthy-sEV and MASLD-sEV, n = 50, Compared with Healthy-sEV group, ∗∗ P < .01; ( M ) ROC curve of OGT in serum sEV, AUC = .7228, P = .0001; ( N ) Correlation analysis of sEV-OGT and serum ALT, r = .2395, P = .0246; ( O ) Correlation analysis of sEV-OGT and serum AST, r = .2493, P = .0171; ( P ) Correlation analysis of sEV-OGT and Fib-4 score (Fib-4 = age × AST/PLT × √ALT), r = .3100, P = .0113.

Article Snippet: After blocking with 5% skim milk for 1 hour to eliminate nonspecific binding, the membrane was incubated overnight at 4°C with primary antibodies: β-actin (ABclonal, AC026, Rabbit), V5-Tag (ABclonal, AE101, Rabbit), TSG101 (Bioworld, BS91381, Rabbit), Calnexin (Bioworld, BS1438, Rabbit), CD9 (Proteintech, 60232-1-Ig, Mouse), CD63 (Abcam, ab271286, Rabbit), Ang-2 (Abcam, ab155106, Rabbit), CD31 (Proteintech, 11265-1-AP, Rabbit), OGT (Proteintech, 11576-2-AP, Rabbit), O-GlcNac (CST, #9875, Mouse), and HNF1α (Proteintech, 22426-1-AP, Rabbit).

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

Journal: RSC Advances

Article Title: Profiling characteristics of plasma exosomal miRNAs across cognitive stages: from normalcy to mild cognitive impairment and Alzheimer's disease †

doi: 10.1039/d5ra02993g

Figure Lengend Snippet: Serum exosomes isolation and identification. (A) Representative morphological structures of exosomes observed by transmission electron microscopy (scale bar: 100 nm). (B) Particle size distribution analysis of exosomes by nanoparticle tracking analysis. (C) Expression of exosome markers CD9 and CD81 detected by nano-flow analysis. The P1 and P2 regions represent the different cell populations and their proportion, respectively. (D) Western blot analysis of Calnexin, CD9, CD81, and TSG101 detected expression on the exosomes.

Article Snippet: The antibodies used were as follows: CD9 (Cat. No. A1703, Abclonal), CD81 (Cat. No. 41779, SAB), Calnexin (Cat. No. ab22595, Abcam), and TSG101 (Cat. No. ab133586, Abcam).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Expressing, Western Blot