Figure 5. Localization of F-actin in MCF-7/MR cells and inhibition of actin polymerization by CytD.

(A) Cells grown for 7 days on glass cover slips were fixed, permeabilized with Triton-X-100 and reacted with anti-ZO-1 antibody (a and d) and with rhodamine phalloidin (b and e) to follow F-actin. The localization of actin and ZO-1 at the EVs surface is shown for EVs formed between two (a–c) and/or multiple attached cells (d–f). Arrows denote the localization of cell-cell attachment zones (belt-like structures). (B) Cells were either untreated (a–c) or treated with CytD (10 µg/ml) for 30 min at 37°C (d–f), washed and reacted as in panel A, to visualize ZO-1 (a and d) and F-actin (b and e). (C) Cells were treated as described in panel B and then stained for ABCG2 (a and d) and F-actin (b and e). (D) Cells were grown in riboflavin (B2)-deficient medium for 48 hr prior to CytD treatment to avoid riboflavin accumulation in EVs. Cells were then washed and transferred to riboflavin-containing medium for an additional 24 hr to examine the riboflavin accumulation capacity (a–c). Untreated cells grown continuously in medium containing (d–f) or lacking riboflavin (g–i) served as controls. Arrows denote the location of EVs. Cells were analyzed using a Zeiss inverted Cell-Observer microscope at a magnification of ×630 (A–C) or ×200 (D). The merged images including DAPI staining (panels A–C), were generated using Cell-Observer software.