Journal: Molecular & Cellular Proteomics : MCP

Article Title: The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions

doi: 10.1016/j.mcpro.2023.100514

Figure Lengend Snippet: Characterization of extracellular vesicle (EVs) subsets, namely, small (S-EVs) or large (L-EVs), isolated from pig seminal plasma (SP) samples (n = 3; three ejaculates per sample; one ejaculate per male pig). A , violin plot displaying the total protein concentration in both SP-EV subsets. The dashed line shows the median and dotted lines the 25 to 75% quartiles. B , representative histogram of particle size distribution of S-EVs and L-EVs assessed by nanoparticle tracking analysis. C , particle size distribution of S-EVs and L-EVs analyzed by dynamic light scattering ( red , S-EVs; blue , L-EVs) in terms of intensity and volume. The black and gray lines represent the average of intensity size distribution of S-EVs and L-EVs, respectively. D , representative images of the morphology of S-EVs and L-EVs assessed by transmission electron microscopy. E , representative histogram of CFSE/CD63/HSP90β/ALB expression in S-EVs and L-EVs assessed by flow cytometry. ALB, albumin; CFSE, carboxyfluorescein succinimidyl ester; CNT, control; HSP90β, heat shock protein 90β.

Article Snippet: The EVs were cytometrically characterized following the International Society of Extracellular Vesicles recommendations (MIFlowCyt-EV, ( )) to identify their enrichment in proteins belonging to the three categories established by MISEV 2018 guidelines ( ): CD63 (Anti-CD63-FITC, Clone REA1055, Miltenyi Biotec) as “Category 1” protein (Transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes); HSP90β (anti-HSP90β-PE, ADI-SPA-844PE-050, Enzo Life Sciences) as “Category 2” protein (Cytosolic proteins recovered in EVs), and albumin (Anti-swine Albumin-FITC, CLFAG16140, Cedarlane) as “Category 3” protein (Major components of non-EVs coisolated structures).

Techniques: Isolation, Clinical Proteomics, Protein Concentration, Transmission Assay, Electron Microscopy, Expressing, Flow Cytometry, Control

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

Journal: Molecular Cancer

Article Title: Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA)

doi: 10.1186/s12943-015-0400-7

Figure Lengend Snippet: Isolation and molecular characterization of exosomes shed from breast cancer cells. a Exosomes were isolated from the conditioned media of control or treated MCF7 cells (100 μM DHA for 24 h) using the Exoquick Reagent, fixed, whole-mounted on coated Formvar grids, negatively stained and visualized by electron microscopy (Hitachi H-7600). Left column, 40,000×; right column 100,000×. Images are representative of three experiments. b Immunogold labeling and electron microscopy of human CD63 (10 nM gold particles) on exosomes isolated from MCF7 cells (100,000×). c Western blot analysis of the exosome marker CD63 (30–60 kDa) in exosomes shed from MCF7 and MDA-MB-231 cells. Exosome lysates were run under non-reducing conditions. d RNA Agilent Bioanalyzer profile of total RNA extracted from control and DHA treated MCF7 cells and their purified exosomes. The peak at 25 nt is a lower marker standard and is indicated on the upper left image. In the exosome preparations the RNA is predominantly <1,000 nucleotides in length, with a large peak noted in the small RNA range of the DHA-treated exosomes and the 28S and 18S ribosomal subunit RNAs (as indicated on upper left image) are notably absent from both treated and untreated exosome preparations

Article Snippet: CD63-GFP (copGFP) levels were measured on BioTek Synergy HT plate reader (BioTek Instruments, Inc. Winooski, VT) and detected by excitation at 485 nm and emission at 528 nm.

Techniques: Isolation, Staining, Electron Microscopy, Labeling, Western Blot, Marker, Purification

Journal: Molecular Cancer

Article Title: Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA)

doi: 10.1186/s12943-015-0400-7

Figure Lengend Snippet: DHA increases exosome secretion from breast cancer cells and reduces tube formation by endothelial cells without affecting VEGF secretion. a Western blot analysis of CD63 expression in exosomes isolated from MCF7 and MDA-MB-231 cells after treatment with DHA. Equal volumes of exosome protein lysate were loaded and SDS-PAGE was run under non-reducing conditions. b CD63-GFP tagged exosomes secreted into cell culture medium from DHA-treated or untreated MCF7 and MDA-MB-231 breast cancer cells were isolated by ultracentrifugation, re-suspended in PBS and the GFP levels were measured by fluorescent spectrometry, * p < 0.0001 using one-way ANOVA ( n = 3). c MCF7 and MDA-MB-231 breast cancer cells were treated with 100 μM DHA for 24 h. The media was removed for exosome isolation and the breast cancer cells that remained after DHA treatment were counted and are expressed as the average cell number per 10 6 cells ( n = 3, error bars = SE). d MCF7 cells were treated with 100 μM DHA for 24 h, the exosomes from the treated and untreated cultures were isolated from the culture media by ultracentrifugation and stained with acridine orange (AO). The AO-labeled exosomes (100 μg) were incubated with EA.hy926 cells (1×10 4 ) seeded on ECMatrix in a 12-well plate for 24 h and imaged with a Perkin Elmer Operetta at 40× magnification, bright field (left) and fluorescence (right), 460 nm excitation and 650 nm emission. e Approximately 1/10 of the exosomes isolated from a confluent 100 mm plate of DHA-treated or untreated MCF7 cells were mixed with 1×10 4 EA.hy926 cells and seeded on ECMatrix in a 96-well plate and incubated for 18 h. Tube formation was visualized with a light microscope ( lower ) and counted ( upper ) *, p < 0.001 using one-way ANOVA ( n = 4). f MCF7 cells were treated with 100 μM DHA, 1 mM clofibrate (CF) or 20 μM troglitazine (Trog) for 24 h and VEGF in the medium was analyzed by ELISA and expressed as pg/ml. *, p < 0.001 using one-way ANOVA ( n = 4-6)

Article Snippet: CD63-GFP (copGFP) levels were measured on BioTek Synergy HT plate reader (BioTek Instruments, Inc. Winooski, VT) and detected by excitation at 485 nm and emission at 528 nm.

Techniques: Western Blot, Expressing, Isolation, SDS Page, Cell Culture, Staining, Labeling, Incubation, Fluorescence, Light Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cancer

Article Title: Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA)

doi: 10.1186/s12943-015-0400-7

Figure Lengend Snippet: Rab27A knockdown in breast cancer cells inhibits DHA-induced exosome transfer. The expression of Rab27A was knocked down in MCF7 CD63-GFP and MDA-MB-231 CD63-GFP breast cancer cell lines by transduction with a lentivirus expressing dsRed2 and two different shRNAs targeting Rab27A. a Bright field ( left ) and fluorescent microscopic images ( right ) of CD63-GFP (green), shRab27A-dsRed2 (red) and Hoescht nuclear stain (blue) (40× magnification). Rab27A protein and mRNA knockdown was confirmed by western blot ( b ) and qRT-PCR ( c ). The fold change in expression levels of Rab27A relative to an endogenous control was calculated using the ΔΔCt method. Data in bar graphs represent mean ± SEM *, p < 0.001 using Student’s t test. d The quantity of exosomes secreted from breast cancer cells was measured by BCA assay and represented as the μg/μl of whole exosome per 10 6 cells, relative to wildtype. Data in bar graphs represent mean ± SEM ( n = 2). e MCF7 and MDA-MB-231 cells expressing CD63 or shRab27A-CD63 were grown in a transwell insert (0.4 μm) and co-cultured with EA.hy926 cells plated on ECMatrix for 24 h. Following treatment of the breast cancer cells in the upper chamber with 100 μM DHA for 24 h the number of branch points formed by the EA.hy926 cells were counted. Data in bar graphs represent mean ± SEM ( n = 3-6) *, p < 0.05; **, p < 0.01 using Student’s t test. f MCF7 cells expressing CD63 or shRab27A-CD63 were grown in a transwell insert (0.4 μm) and co-cultured with EA.hy926 cells plated in a 24-well plate. Following treatment of the breast cancer cells in the upper chamber with 100 μM DHA for 24 h total RNA was extracted from the EA.hy926 cells, reverse transcribed and expression of miR-23b and miR-320b was measured by qRT-PCR. The fold change in expression levels was calculated using the ΔΔCt method. Fold-change values are normalized to control sample and expressed relative to no breast cancer cells (−BC). Data in bar graphs represent mean ± SEM ( n = 3) *, p ≤ 0.05; **, p < 0.001 using Student’s t test

Article Snippet: CD63-GFP (copGFP) levels were measured on BioTek Synergy HT plate reader (BioTek Instruments, Inc. Winooski, VT) and detected by excitation at 485 nm and emission at 528 nm.

Techniques: Expressing, Transduction, Staining, Western Blot, Quantitative RT-PCR, BIA-KA, Cell Culture

Journal: Frontiers in Pharmacology

Article Title: Celecoxib ameliorates diabetic sarcopenia by inhibiting inflammation, stress response, mitochondrial dysfunction, and subsequent activation of the protein degradation systems

doi: 10.3389/fphar.2024.1344276

Figure Lengend Snippet: Celecoxib inhibited inflammation during diabetes-induced muscle atrophy. (A) Hematoxylin-eosin (H&E) staining of tibialis anterior, scale bar = 20 μm; and CD68 immunofluorescence staining of tibialis anterior, scale bar = 100 μm; (B–C) The levels of CD68 and CD86 were tested in tibialis anterior by Western blotting, and their quantitative analysis; (D–E) Western blotting analysis of the levels COX2, PGE2, IL-6, TNF-α, and the phosphorylation levels of NF-κB and Stat3 in tibialis anterior, and quantitative analysis; (F–G) The levels and grayscale value statistics of NLRP3, Caspase1, IL-1β of NLRP3 inflammasome pathway in tibialis anterior. Data are presented as mean ± SD, n ≥ 3; **, p < 0.01 DM versus control; ***, p < 0.001 DM versus control; #, p < 0.01 DM + CXB versus DM; ##, p < 0.01 DM + CXB versus DM; ###, p < 0.001 DM + CXB versus DM. DM: diabetes mellitus; CXB: celecoxib.

Article Snippet: The antibodies included: β-Tubulin (#ab6046), Cox2 (#ab15191), IL-1β(#ab254360), Nox2(#ab129068), Nox4(#ab109225), GPX1(#ab22604), Nrf2(#ab137550), Fbx32(#ab168372), MHC(#ab91506), Beclin1(#ab207612), ATG7(#ab133528), PGC1α(#ab191838), IL-6 (#ab9731), Sirt1 (#ab110304), BNIP3 (#ab10433), Goat Anti-Rabbit IgG H&L (HRP) (#ab205718), Goat Anti-Mouse IgG H&L (HRP) (#ab205719) from Abcam; CD68 (#38005), MuRF1 (#38580) from SAB; CD86 (#26903-1-AP), PTGES2 (#10881-1-AP), TNF-α (#60291-1-lg) from Proteintech; p-NF-κB (#3033S), p-Stat3 (Tyr705, #9145S), NLRP3(#30835S), Caspase1 (E9R2D, #83383S), Perk (#3192S), EIF-2α (#9722S), p-EIF-2α (#9721S), ATF-4 (#11815S), Chop (#2895S), Foxo3a (#12829S) from Cell Signaling Technology; p-Perk (#PA5-40294) from Invitrogen; LC3II (#M186-3) from MBL.

Techniques: Staining, Immunofluorescence, Western Blot, Phospho-proteomics, Control

Journal: Cancer Cell International

Article Title: mir-605-3p prevents liver premetastatic niche formation by inhibiting angiogenesis via decreasing exosomal nos3 release in gastric cancer

doi: 10.1186/s12935-024-03359-5

Figure Lengend Snippet: miR-605-3p suppresses GC angiogenesis by inhibiting the release of exosomes. (a) Electron microscopy scans of exosomes derived from SGC-7901/miR-NC, SGC-7901/miR-605-3p mimic, AGS/miR-NC, and AGS/miR-605-3p inhibitor cells (scale bar, 100 nm). (b) Characterization and quantitation of vesicles derived from the same number of SGC-7901/miR-NC, SGC-7901/miR-605-3p mimic, AGS/miR-NC, and AGS/miR-605-3p inhibitor cells as assessed by NTA. (c) WB was performed to evaluate the expression of the exosomal markers CD63 and TSG101 and the marker calnexin of lysates from the same number of GC cells. (d) Fluorescence of HUVECs incubated for 48 h with PKH67-labelled exosomes derived from SGC-7901/miR-NC and SGC-7901/miR-605-3p mimic cells (scale bar, 25 μm). (e) Effect of exosomes derived from the same number of SGC-7901/miR-NC, SGC-7901/miR-605-3p mimic, AGS/miR-NC, and AGS/miR-605-3p inhibitor cells on tube formation ability (scale bar, 100 μm). ** P < 0.01, *** P < 0.001

Article Snippet: HUVECs seeded onto a cell climbing tablet in a 24-well plate were fixed, permeabilized, blocked, and incubated overnight at 4 °C with primary antibodies targeting the following proteins: Tubulin (Proteintech, 11224-1-AP, 1:100) and CD63 (Abcam, ab217345, 1:100).

Techniques: Electron Microscopy, Derivative Assay, Quantitation Assay, Expressing, Marker, Fluorescence, Incubation

Journal: Aging (Albany NY)

Article Title: LncRNA PVT1 promotes exosome secretion through YKT6, RAB7, and VAMP3 in pancreatic cancer

doi: 10.18632/aging.103268

Figure Lengend Snippet: PVT1 promotes the movement of MVBs towards the plasma membrane. ( A ) Analysis of CD63 (red) in PVT1-overexpressing HS766T cells, as determined by confocal microscope. Nuclei were labeled with DAPI (blue). ( B ) Analysis of YKT6 (green) and CD63 (red) in PVT1-overexpressing HS766T cells, as determined by confocal microscope. ( C ) Exosomes in PVT1-overexpressing HS766T cells, as determined by electron microscope.

Article Snippet: The primary antibodies used were as following: ras-related protein Rab-7 (RAB7) (1:100), CD63 (1:200), YKT6 v-SNARE homolog (YKT6) (1:100), and vesicle-associated membrane protein 3 (VAMP3) (1:100) (Applied Biological Materials Inc., Richmond, BC, Canada).

Techniques: Clinical Proteomics, Membrane, Microscopy, Labeling

Journal: Aging (Albany NY)

Article Title: LncRNA PVT1 promotes exosome secretion through YKT6, RAB7, and VAMP3 in pancreatic cancer

doi: 10.18632/aging.103268

Figure Lengend Snippet: PVT1 affects the expression and localization of RAB7 in HS766T cells. ( A ) The mRNA expression of Rab GTPases genes in PVT1-overexpressing HS766T cells. ( B ) The protein expression of RAB7 in PVT1-overexpressing HS766T cells. ( C ) Analysis of RAB7 (green) and CD63 (red) in PVT1-overexpressing HS766T cells, as determined by confocal microscope. ( D ) The interaction between PVT1 and RAB7, as determined by RIP assay and qRT-PCR. ( E ) The correlation between PVT1 and RAB7, as determined by pull-down assay. ( F ) The knockdown efficiency of si-RAB7 in HS766T cells. ( G ) The concentration of exosome derived from HS766T cells with overexpression of PVT1 and knockdown of RAB7. * P < 0.05, data are expressed as the mean ± SD.

Article Snippet: The primary antibodies used were as following: ras-related protein Rab-7 (RAB7) (1:100), CD63 (1:200), YKT6 v-SNARE homolog (YKT6) (1:100), and vesicle-associated membrane protein 3 (VAMP3) (1:100) (Applied Biological Materials Inc., Richmond, BC, Canada).

Techniques: Expressing, Microscopy, Quantitative RT-PCR, Pull Down Assay, Knockdown, Concentration Assay, Derivative Assay, Over Expression

Journal: Cancer Medicine

Article Title: Exosome‐mediated delivery of miR‐204‐5p inhibits tumor growth and chemoresistance

doi: 10.1002/cam4.3248

Figure Lengend Snippet: Characteristics of exosomes derived from miR‐204‐5p‐overexpressing HEK293T cells. A, The markers of exosomes (CD63 and Flotillin‐2) were detected in HEK293T cells and exosomes by Western blot. B, The transmission electron micrograph showed roundshaped vesicles with bilayered membranes ranging from 100 nm to 150 nm in diameter released by HEK293T cells. Scale bar = 200 nm. C, 293T EXOs size distribution was measured by Zetasizer. D, Real‐time qRT‐PCR revealed that the level of miR‐204‐5p was higher in 293T‐204 cells and miR‐204 EXO than miR‐204‐3p. *** P < .001. Shown are mean ± SEM from three independent experiments

Article Snippet: The protein samples of cells and exosomes were detected with antibodies against CD63 (1:1000; BOSTER), Flotillin‐2 (1:500; Santa Cruz), RAB22A (1:1000; Proteintech), Bcl2 (1:1000; Santa Cruz) and β‐actin (1:2000; Thermo) as we previously described.,

Techniques: Derivative Assay, Western Blot, Transmission Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

doi: 10.3390/ijms22031105

Figure Lengend Snippet: ( A ) Western Blot analysis of proteins present (Hsp90, CD63 and TSG101) or absent (calnexin) in exosomes and abundant in bovine milk (β-casein). Protein evaluation in: bovine skimmed milk (SM); exosomes obtained from SM by one (1U) or two (2U) ultracentrifugation steps; and the cellular fraction (CF). Equal amount of protein was loaded. Elution protein profile (F.1, F.8 to F.40) of bovine exosomes isolated by 1U followed by SEC (1U + SEC) ( B ) or 2U + SEC ( C ). Protein concentration (mg/mL) was estimated by the BCA assay. WB of SEC elution fractions from exosomes isolated by 1U + SEC ( D ) or 2U + SEC ( E ). Evaluation of Hsp90, CD63, TSG101, Calnexin and β-casein levels in each fraction (F.1, F.8 to F.40). Mimic hsa-miRNA-148a-3p elution profile (relative expression) of exosomes isolated from skimmed milk by U + T + SEC ( F ) or U + T + U + SEC ( G ). Mw: Molecular weight marker (Bio-Rad).

Article Snippet: Membranes were then incubated with appropriate primary antibodies: anti-Hsp90 (610418, BD, Madrid, Spain), anti-CD63 (bs-1523R, BIOSS, Woburn, MA, USA), anti-TSG101 (A303-506A, Bethyl, Montgomery, TX, USA), anti-calnexin (A303-694A, Bethyl) or anti-β-casein (ab112595, abcam, Cambridge, UK) at 4 °C (overnight).

Techniques: Western Blot, Isolation, Protein Concentration, BIA-KA, Expressing, Molecular Weight, Marker