Fig. 4
Analysis of the EV cargo proteome. (A, B) PCA of statistically significant protein expression differences between EVs enriched by the CD81-specific (A) or the CTB-specific (B) method in the supernatant of MDA-MB-231 cells stimulated with vehicle (Ctrl) or 5 ng/mL TGF-β1 for 120 h. Three independent biological repeats were analyzed per condition. (C, D) Venn diagram summarizing the total number of significant and differentially expressed proteins in at least two biological conditions shown in panel A and B. (E, F) Volcano plot of the significant (log2(p-value); y-axis) and differentially expressed (log2(fold-change/FC); x-axis) common proteins between the same two biological conditions shown in panels A-D respectively, expressed as proteins enriched (Up) in the EVs of control or TGF-β1-stimulated cells. Selected protein IDs are shown. Dotted lines indicate the filtering levels along each axis. (G, H) Tables of highly significant gene ontology (GO) and Reactome (REAC) terms represented in the two biological conditions analyzed using the CD81-specific EV isolation method, indicating the term name and associated false discovery rate (FDR) q-value. (I) RT-qPCR analysis of the indicated mRNAs, selected based on the corresponding proteins that scored significantly in the proteomic analysis of MDA-MB-231 cells after stimulation with 5 ng/mL TGF-β1 for 0 (Ctrl), 48 and 120 h. (J) Active and total TGF-β1 concentration was measured through ELISA-sandwich in EVs derived from control cells (VSFCtrl) or cells stimulated with 5 ng/mL TGF-β1 for 120 h (VSF+ TGF−β1). TGF-β1 concentrations are presented based on two-way ANOVA, followed by multiple paired comparisons conducted by means of Bonferroni’s post-test method: **p ≤ 0.01; ****p ≤ 0.0001