mtDNA is protected within EVs. (a) EVs were purified from MDA-MB-231 cell-exposed medium by differential centrifugation and then incubated in the presence and absence of DNase, which had been immobilized on agarose beads. Following removal of the DNase-conjugated beads by centrifugation, the quantity of mtDNA was determined relative to EVs not treated with DNase using qPCR with primers complementary to sequences within the indicated mitochondrial genes and the β-globin nuclear gene. Bars represent the fold change relative to control. Values are the mean ± SEM. (b) MDA-MB-231 cells were transfected with siRNAs targeting CD63 (siCD63) or a nontargeting siRNA (siNT) or were left untransfected. Cells were then incubated for 48 h in the absence (Ctrl; NT and siCD63) or presence of 0.3 µM LY95, and medium was collected over this period. EVs were purified from the medium by differential centrifugation as above, and half of the sample was treated with DNase-conjugated beads. The mtDNA content of these samples was determined using qPCR with primers complementary to a sequence within the mitochondrial ND1 gene. Values represent the mean fold change relative to control from three independent experiments. Error bars represent SEM. **, P < 0.002; Mann-Whitney U test.