Fig. 3

Evaluation of exosome uptake by recipient cells. a Confocal microscopy visualization of untreated (top) and exosome fused (bottom) Me1007 cell line. Me1007/miR-222 donor cells were labeled with the fluorescent BODIPY^® FL C16 fatty acid molecule, which being incorporated into the EXO membranes allowed visualizing these vesicles. Me1007 recipient cells were stained for phalloidin (Alexa Fluor 647-red) and nuclei counterstained with Hoechst. Bodipy C16-labelled exosomes appear as internalized green dots. Scale bar 10 μm. b The uptake of miR-222-containing exosomes by the acceptor Me1007 and Me1402/R melanomas was quantified by specific qRT-PCR (miR-222/RNU6B). Data are cumulative of three independent experiments. Differences in miR-222 expression were evaluated using analysis of variance (ANOVA) followed by a Newman–Keuls post hoc test. Significance was accepted when the p value was <0.05. c The downregulation of p27Kip1, a direct target of miR-222, by EXO/miR-222 was evaluated by qRT-PCR. d Cell cycle analysis, showing the miR-222-dependent early onset of DNA synthesis, was performed on synchronized cells 2 h after exosome internalization. e, f EXO-dependent effects on invasion (left panels) and chemotaxis (right panels) in Me1007 and Me1402/R melanoma cell lines. MiR-222-transduced cells were included as a positive control. Data are representative of two independent experiments. *p < 0.05; **p < 0.01