Inhibition of mitophagy does not oppose release of mtDNA-containing EVs. (a–d) MDA-MB-231 cells were transfected with siRNAs targeting ATG5 and ATG7 (siATG5/7; a and b), FIP200 (siFIP200; c and d), or a nontargeting control (siNT). Conditioned medium was collected from transfected cells over a 48-h period. EVs were purified from conditioned medium using differential centrifugation and analyzed by nanoparticle tracking. mtDNA was determined using qPCR with primers complementary to a sequence within the mitochondrial ND2 gene. Values represent the mean of three independent experiments; dotted line and error bars represent SEM. Cellular levels of ATG5/7 (a), FIP200 (c), and LC3B (a and c) were determined using Western blotting with actin or vinculin as a loading control. In a and c, cells were nutrient starved in Earle’s balanced salt solution (EBSS) where indicated before lysis.