Journal: iScience

Article Title: Effects of lysate/tissue storage at −80°C on subsequently extracted EVs of epithelial ovarian cancer tissue origins

doi: 10.1016/j.isci.2023.106521

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CD81 , System Biosciences , Cat#EXOAB-CD81A-1; RRID: AB_2819191.

Techniques: Recombinant, Software

Journal: International Journal of Molecular Sciences

Article Title: Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells

doi: 10.3390/ijms25116175

Figure Lengend Snippet: Characterization of small and large EVs released by HT1080-derived 2FTGH cells after autophagy stimulation. ( A ) Representative TEM images of large EVs (p15, lEVs) and small EVs (p120, sEVs) fractions obtained from 2FTGH cells, untreated or treated with HBSS for 4 h at 37 °C (scale bar: 200 nm). ( B ) The size distribution of large EVs (p15, lEVs) and small EVs (p120, sEVs) from HBSS-treated (4 h at 37 °C) or untreated cells was obtained by measuring at least 60 vesicles for each sample in 5 randomly selected microscopic fields. ( C ) Immunoblots of large EVs (lEVs) and small EVs (sEVs) fractions from cells, untreated or treated with HBSS, using anti-CD81, anti-ALIX, anti-Annexin A1 (ANXA1), and anti-GM130 antibodies. A representative experiment among 3 is shown.

Article Snippet: The membranes were probed with rabbit anti-CD63 antibodies (Abcam, ab216130), rabbit anti-CD81 (Abcam, ab155760), anti-Annexin A1 (Santa Cruz Biotechnology, sc-12740) antibodies, rabbit anti-ALIX (Abcam, AB22555), anti-GM130 (Santa Cruz, SC55591), rabbit anti-ERLIN1 antibodies (Thermo Fisher Scientific, Waltham, MA, USA, PA5-17102), mouse anti-LC3-II antibodies (Abcam, ab243506), or mouse anti-GD3 R24 antibodies (Abcam, ab11779).

Techniques: Derivative Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells

doi: 10.3390/ijms25116175

Figure Lengend Snippet: Analysis of small EVs by 6–30% iodixanol gradient density. ( A ) Immunoblot analysis of iodixanol density gradient-purified fractions obtained from HT1080-derived 2FTGH cells, untreated or treated with HBSS for 4 h at 37 °C. Twelve fractions, corresponding to low-density fractions (3–5) indicative of small EVs and high-density fractions (8–12) enriched in non-vesicular (NV) components, were probed with mouse anti-LC3-II antibodies, with rabbit anti-ERLIN1 antibodies, with rabbit anti-CD63 antibodies, with rabbit anti-CD81 antibodies, or with rabbit anti-fibronectin. The observed MW may vary in the range of 25–65 (predicted MW: 26 kDa), depending on post-translational modifications, post-translational cleavages, relative charges, and other experimental factors, including the unreducing condition. A representative experiment among 3 is shown. ( B ) The bar graph shows densitometric analysis. Results represent the mean ± SD. ** p < 0.005. ( C ) Representative TEM images of iodixanol density gradient-purified fractions 5 (F5) and 11 (F11) obtained from 2FTGH cells, untreated or treated with HBSS for 4 h at 37 °C (scale bar: 200 nm).

Article Snippet: The membranes were probed with rabbit anti-CD63 antibodies (Abcam, ab216130), rabbit anti-CD81 (Abcam, ab155760), anti-Annexin A1 (Santa Cruz Biotechnology, sc-12740) antibodies, rabbit anti-ALIX (Abcam, AB22555), anti-GM130 (Santa Cruz, SC55591), rabbit anti-ERLIN1 antibodies (Thermo Fisher Scientific, Waltham, MA, USA, PA5-17102), mouse anti-LC3-II antibodies (Abcam, ab243506), or mouse anti-GD3 R24 antibodies (Abcam, ab11779).

Techniques: Western Blot, Purification, Derivative Assay

Journal: bioRxiv

Article Title: Homosalate boosts the release of tumor-derived Extracellular Vesicles with anti-anoikis properties

doi: 10.1101/2021.10.25.465564

Figure Lengend Snippet: A) Quantification of cell viability after Homosalate treatment in MDA-MB-231 parental cells. DMSO or Homosalate treated cells were counted after collection of conditioned media, using Trypan Blue as a reporter of cell death. Cell viability is expressed in percentage. Data from five independent experiments are shown. B) Quantification of EVs induced by treatment with Homosalate. Left panel: one representative graph showing particle concentration/cm 3 versus particle size measured by NTA in inputs (total conditioned medium) and F7-11 (EV-rich SEC fractions) from 27*10^6 DMSO or Homosalate treated cells. Right panel: Graphs show total particle number secreted from 27*10^6 cells measured by NTA in DMSO or Homosalate treated cells for inputs and SEC F7-11, from five independent experiments. Paired parametric t-test p=0.006; p=0.004. C) Representative TEM images showing (CD63+) or (CD9+) EVs in F7-11 released by 2.7*10^6 DMSO or Homosalate treated cells. Arrowheads indicate EVs positive for CD63 staining (above) or CD9 staining (below). Graphs show quantification of the number (=nb) of (CD63+EVs) + (CD9+ EVs) per μm 2 . Scale bar 0.5μm. Data from two independent experiments are shown, each dot represents EVs counted in one field (DMSO: 20 dots for replicate 1, 18 dots for replicate 2; Homosalate: 20 dots for replicate 1, 18 dots for replicate 2). Mann-Whitney test p=0.003. D) Western Blot analysis of EV markers SLC3A2/CD98, CD63, Syntenin, CD81 and CD9 released by cells treated with DMSO or Homosalate after gel loading with same number of secreting cells. Gapdh was used as normalizer for cell lysates (CL). CL from the equivalent of 200 000 cells were loaded, F7-11 from the equivalent of 2.7*10^6 secreting cells were loaded. Graphs show protein signal quantifications normalized first on Gapdh and then on DMSO for CL or normalized on DMSO for F7-11. Data from three independent experiments are shown. E) Western Blot analysis of EV markers SLC3A2/CD98, CD63, Syntenin, CD81 and CD9 released by cells treated with DMSO or Homosalate, after gel loading with same numbers of particles. Gapdh was used as normalizer for CL. CL from the equivalent of 200 000 cells or an amount corresponding to 4*10^8 particles for F7-11 were loaded. Graphs show protein signal quantifications normalized first on Gapdh and then on DMSO for CL or normalized on DMSO for F7-11. Data from three independent experiments are shown. F) Western Blot analysis of EVs immunoprecipitated from F7-11 and schemes of the EVs recovered. The equivalent of 1*10^9 particles in F7-11 were immunoprecipitated with anti-CD63- or anti-CD9-coated beads (IP) and loaded side-by-side with the corresponding unbound EVs contained in the flow-through (FT). Blots were incubated with SLC3A2/CD98, CD63 or CD9 antibodies. Percent of immunoprecipitation displayed in the right panel graph was calculated as signal for SLC3A2/CD98, CD63, CD9 present in CD63 or CD9 IP divided by the total signal for the protein in the same IP: IP/(IP+FT). Data from three independent experiments are shown.

Article Snippet: Membrane were incubated with the following antibodies: mouse anti-human SLC3A2/CD98 1/3000 (clone 2B10F5 ProteinTech), mouse anti-human CD63 1/1000 (clone H5C6, BD Bioscience), mouse anti-human CD9 1/1000 (clone MM2/57, Millipore), mouse anti-human CD81 1:1000 (clone TS81, Diaclone), rabbit anti-human 14-3-3 1/1000 (EPR6380, GeneTex), rabbit anti-human Lamp1 1/1000 (clone EPR4204, GeneTex), mouse anti-Gapdh (clone 1E6D9, ProteinTech).

Techniques: Concentration Assay, Staining, MANN-WHITNEY, Western Blot, Immunoprecipitation, Incubation

Journal: bioRxiv

Article Title: Homosalate boosts the release of tumor-derived Extracellular Vesicles with anti-anoikis properties

doi: 10.1101/2021.10.25.465564

Figure Lengend Snippet: A) Quantification of cell viability after Homosalate and Bafilomycin A1 treatment in MDA-MB-231 parental cells. DMSO, Homosalate or Bafilomycin A1 treated cells were counted after conditioned media collection, using Trypan Blue as a reporter of cell death. Cell viability is expressed in percentage. Data from five independent experiments are shown. B) Quantification of EVs induced by treatment with Homosalate and Bafilomycin A1 in MDA-MB-231 parental cells. Left panel: Graphs show particle concentration/cm 3 versus particle size measured by NTA in F7-11 (EV-rich SEC fractions) from 50*10^6 DMSO, Homosalate or Bafilomycin A1 treated cells. Right panel: Graphs show total particle number secreted from 50*10^6 cells measured by NTA in DMSO, Homosalate or Bafilomycin A1 treated cells for SEC F7-11. Data from five independent experiments are shown. Paired parametric t-test p=0.04; p=0.002. C) Western Blot analysis of EV markers SLC3A2/CD98, CD9, CD81, Syntenin, CD63 and Lamp-1 released after treatment with DMSO, Homosalate or Bafilomycin A1 after gel loading with same number of particles. CL from the equivalent of 200 000 cells or an amount corresponding to 4*10^8 particles from F7-11 were loaded. Graphs show protein signal quantifications normalized to DMSO for F7-11 after treatment with Homosalate (above) or Bafilomycin A1 (below). Data from five independent experiments are shown. D) Immunofluorescence of SLC3A2/CD98, CD63 and CD9 in MDA-MB-231 treated with DMSO, Homosalate or Bafilomycin A1. E) Graphs show Mander’s correlation coefficients for CD98-CD9, CD63-CD9 or CD63-CD98 co-localization expressed as percentage. Ordinary one way Anova, multiple comparison test CD98-CD9: ns or p=0.001; CD63-CD9: ns or p=0.0008; CD63-CD98 p=0.0001. Data from three independent experiments are shown, each dot represents one counted cell (DMSO: 25 dots for replicate 1, 10 dots for replicate 2, 19 dots for replicate 3; Homosalate: 25 dots for replicate 1, 12 dots for replicate 2, 21 dots for replicate 3; Bafilomycin A1: 21 dots for replicate 1, 16 dots for replicate 2, 22 dots for replicate 3).

Article Snippet: Membrane were incubated with the following antibodies: mouse anti-human SLC3A2/CD98 1/3000 (clone 2B10F5 ProteinTech), mouse anti-human CD63 1/1000 (clone H5C6, BD Bioscience), mouse anti-human CD9 1/1000 (clone MM2/57, Millipore), mouse anti-human CD81 1:1000 (clone TS81, Diaclone), rabbit anti-human 14-3-3 1/1000 (EPR6380, GeneTex), rabbit anti-human Lamp1 1/1000 (clone EPR4204, GeneTex), mouse anti-Gapdh (clone 1E6D9, ProteinTech).

Techniques: Concentration Assay, Western Blot, Immunofluorescence