Fig 1: Expression of EV and ovarian cancer biomarkers in EV fractions. A–G: Full-panel blots of data shown in Figure 2 and additional Western blot analysis of whole cell extract (WCE) from COV413A cells and size-exclusion chromatography (SEC) purified fractions from COV413A conditioned media (EV1 to EV6) using antibodies against EV surface biomarkers CD9 (A), CD81 (B), and CD63 (C); EV cargo biomarkers flotillin (D) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; E) demonstrate the EVs were intact during enrichment; and non-EV internal membrane proteins mitochondrial import receptor subunit TOM20 homolog (TOM20) (F) and ribosomal protein S3 (RPS3) (G) demonstrate the EV fractions are the result of EV biogenesis and do not include intracellular vesicles. H: Total protein staining (Coomassie) was used to validate SEC separation of the EV fractions from the soluble protein component and was confirmed by measuring the protein concentration in each fraction (Figure 2). I–K: Full-panel blots of data shown in Figure 2 demonstrating abundance of bone marrow stromal antigen-2 (BST2; I), folate receptor-α (FOLR1; J), and mucin-1 (MUC1)-C (K) in COV413A EVs. L: Representative molecular weight markers with indicated observed molecular weights are shown as a reference. Source data: uncropped blot images. A–G: Western blot analysis of WCE from COV413A cells and SEC purified fractions from COV413A conditioned media (EV1 to EV6) using antibodies against EV surface biomarkers CD9 (A), CD81 (B), and CD63 (C); EV cargo biomarkers flotillin (D) and GAPDH (E) to demonstrate the EVs were intact EVs during enrichment; and non-EV internal membrane proteins Tom20 (F) and RPS3 (G) to demonstrate the EV fractions are the result of EV biogenesis and do not include intracellular vesicles. H: Total protein staining (Coomassie stain) was used to validate SEC separation of the EV fractions from the soluble protein component and was confirmed by measuring the protein concentration in each fraction as shown in Figure 2. I–K: Full-panel blots of data shown in Figure 2 demonstrating abundance of BST2 (I), FOLR1 (J), and MUC1-C (K) in COV413A EVs. Biotinylated molecular weight markers were detected using anti-biotin horseradish peroxidase antibody and are indicated on each panel.
Fig 2: Characterization of human cancer cell line extracellular vesicles. Size-exclusion chromatography (SEC)–enriched EV fractions from human ovarian cancer cell line (COV413A) conditioned media were characterized according to minimal information for studies of extracellular vesicles guidelines. A: Nanoparticle tracking analysis (particle count; greenline) and protein quantification (protein; pinkline) of each fraction collected from the SEC column demonstrating the bulk of the nanoparticles elute in EV1 to EV3, distinct from the bulk of soluble protein (EV6 to EV8). The first fraction (Pre-EV) is the sum of the void and wash volumes of the column, which generally contains larger particles, such as lipoproteins, which are not observed in cell line conditioned media. The purified collection volume (PCV), fractions EV1 to EV3, containing the EVs used in downstream assay steps are highlighted. B: Transmission electron microscopy of EVs derived from the total PCV (pooled fractions EV1 to EV3) from COV413A conditioned media at two different magnifications demonstrates intact EVs ranging from approximately 40 to 300 nm. C: Particle size distribution of pooled SEC fractions EV1 to EV3 demonstrating particles consistent with the size range of small EVs ranging from approximately 40 to 400 nm. D: Western blot analysis of each of the initial fractions from the SEC column (Pre-EV, EV1 to EV6) from COV413A conditioned media demonstrating the presence of known EV surface biomarkers (CD9, CD63, and CD81), as well as bone marrow stromal antigen-2 (BST2), folate receptor-α (FOLR1), and mucin-1 (MUC1). Note that an antibody detecting the cleaved fragment of MUC1 (MUC1-C) was used to facilitate immunoblotting because of the high molecular weight of MUC1 (250 to 500 kDa). Whole cell extracts (WCEs; 10 μg total protein per lane) from COV413A were analyzed in parallel as a control for protein expression and biotinylated molecular weight (Mol. Wt.) markers were used to determine apparent molecular mass. D: Source data: uncropped images from the Western blot analyses shown in Figure 2. Immunoblot analysis of each of the initial fractions from COV413A cell line conditioned media concentrated and purified by SEC, as described in Materials and Methods. Size exclusion fractions (Pre-EV, EV1 to EV6) were evaluated for the presence of known EV surface biomarkers CD9, CD81, CD63, as well as BST2, FOLR1, and MUC1. Note that an antibody detecting MUC1-C was used to facilitate immunoblotting because of the high molecular weight of MUC1 (250 to 500 kDa). WCEs; (10 μg total protein per lane) from COV413A were analyzed in parallel as a control for protein expression, and biotinylated Mol. Wt. markers were used to indicate the apparent molecular mass. Scale bars: 500 nm (B, left panel); 200 nm (B, right panel).
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