Fig. 5

Analysis of EV content. A EV morphology was determined by Transmission Electron Microscopy (TEM). Scale bar = 100 nm. B, C The total amount of extracellular proteins released by A2780 and A2780 CIS cells normalized on total cell proteins and without normalization. D EVs were purified by ultracentrifugation and RAB7 content in cell lysates and EVs were evaluated by Western blot analysis. The purity of EVs was ascertained according to the ISEV guidelines by looking at different negative and positive markers as indicated. E-H Expression levels of CD81, CD9, CD63, AND RAB7 were determined in cellular lysates and in EVs by densitometry normalizing against β-Actin. I-J A2780 CIS were treated with GW4869 EV secretion inhibitor and intracellular levels of RAB7 and CD81 (as a positive control of inhibition) were measured by western blotting analysis normalizing against HSP90. K-P EVs were purified by immunoisolation. Positive and negative controls were used to verify the purity of EVs. Mitochondrial protein expression was evaluated in EVs by Western blot and the relative abundance of RAB7, ATP5A, NDUFS3, SDHA, and SDHB was determined through densitometric analysis normalizing against vinculin. Q Total DNA was extracted from A2780 cells and A2780 CIS EVs. MitoALL resequencing kit was used to analyze mitochondrial DNA (mtDNA) contained in EVs. Mock 1 and Mock 2 represent two conditions of PCR purity control in which PCR mix with amplicon 19 and 21 primers were used as controls. Data in graphs represent the mean ± SEM of at least three independent experiments. * p < 0.05; ** p < 0.01; ***p < 0.001