Involvement of microtubules in the formation of EVs in MCF-7/MR cells and inhibition of microtubule polymerization by nocodazole.

MCF-7/MR cells were either untreated (A–C and G–I) or treated with nocodazole (33 µM) for 1 hr at 37°C (D–F and J–L). Microtubules were visualized using mouse anti β-tubulin antibody followed by incubation with FITC-conjugated donkey anti-mouse IgG (B, E, H and K). Cells were co-reacted with antibodies to ABCG2 (BXP-53, A and D) or ERM (G and J), processed and analyzed as in Fig. 5 legend. Quantification of ABCG2 and ERM protein expression on the surface of EVs prior to and following treatment with nocodazole (M); random fields stained as in upper panels were photographed using the same exposure conditions for untreated and nocodazole-treated cells. The surface area of EVs and its relative fluorescence intensity were estimated using the AxioVision program. The fluorescence at the EVs surface indicated protein levels at the EVs membrane. A total of 100 EVs were analyzed for each examined protein. Bars represent SD.