mtDNA is required for EVs to evoke invasive responses. (a and b) Cells with reduced mtDNA content (Rho0) were generated by incubation of MDA-MB-231 cells with ethidium bromide for 5 d. EVs were collected from control (Ctrl.) and Rho0 cells over a 48-h period and analyzed to determine their number and size distribution (a) and nuclear and mtDNA content (b) as in Figs. 1 a and 2 d, respectively. (c and d) EVs from control (Crtl.) and Rho0 cells were then incubated with glutamine-starved cells, and the ability of these recipient cells to degrade collagen (c) and to extend invasive protrusions (d) was determined as in Fig. 6, c and d, respectively. Bar, 20 µm; bar in zoom, 2 µm. Values are mean ± SEM; n = 3 independent experiments. **, P < 0.002; ***, P < 0.001; ANOVA (one-way ANOVA for c; two-way ANOVA for d) with Dunn’s multiple comparison test.