Figure 2. Mitochondrial proteins and DNA... | Rockefeller University Press

Figure 2.

$Mitochondrial proteins and DNA are packaged into EVs. (a) MDA-MB-231 cells were transfected with siRNAs targeting Rab27a/b (siRab27), CD63 (siCD63), or nontargeting siRNA (siNT) and incubated for 5 d in the presence (LY95) or absence (Ctrl) of LY95 (0.3 µM). Oxygen consumption was determined using the Seahorse XFe96 Extracellular Flux Analyzer. Sequential treatment with oligomycin, FCCP, and rotenone/antimycin A (Rot+AntA) was performed as indicated. Values represent the mean ± SEM of three independent experiments. (b) Cells were incubated in the presence of LY95 (0.3 µM) or vehicle control (Ctrl) for 48 h. EVs were isolated from media by differential centrifugation and analyzed by Western blotting for VDAC, cyclophilin D, and GLUD1. (c) Mitochondria (mito.) were purified from cells and EVs. mtDNA was extracted from these preparations and analyzed by long-range PCR. The expected migration position of the mitochondrial nucleoid (∼16 × 106 D) is indicated. (d) EVs were purified by differential centrifugation and incubated in the presence and absence of DNase immobilized to agarose beads. mtDNA and nuclear DNA were determined by qPCR using primers complementary to sequences within the mitochondrial genes (COX1, CYTB, n = 5 independent experiments; or ND1, n = 3 independent experiments) or the nuclear β-globin genes. Values are mean ± SEM. *, P < 0.05; paired t test. (e) Cells were transfected with siRNAs targeting CD63 (siCD63) or a nontargeting siRNA (siNT). Cells were then incubated for 48 h in the absence (Ctrl; NT and siCD63) or presence of 0.3 µM LY95. EVs were purified from the medium by differential centrifugation, and mtDNA content of these was determined using qPCR with primers recognizing the mitochondrial COX1 gene. Values are mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.002; ***, P < 0.001; Mann-Whitney U test. (f) Media collected in the absence (Ctrl) and presence of 0.3 µM LY95 were subjected to differential centrifugation. Differential centrifugation pellets were overlaid with a sucrose density gradient (2–0.4 M sucrose) and centrifuged at 200,000 g overnight. Gradients were eluted, and fractions were collected for analysis by qPCR for the mitochondrial ND1 gene and by Western blotting for CD63. Values represent mean ± SEM, n = 5 independent experiments. *, P < 0.05; Mann-Whitney U test.$

Mitochondrial proteins and DNA are packaged into EVs. (a) MDA-MB-231 cells were transfected with siRNAs targeting Rab27a/b (siRab27), CD63 (siCD63), or nontargeting siRNA (siNT) and incubated for 5 d in the presence (LY95) or absence (Ctrl) of LY95 (0.3 µM). Oxygen consumption was determined using the Seahorse XF^e96 Extracellular Flux Analyzer. Sequential treatment with oligomycin, FCCP, and rotenone/antimycin A (Rot+AntA) was performed as indicated. Values represent the mean ± SEM of three independent experiments. (b) Cells were incubated in the presence of LY95 (0.3 µM) or vehicle control (Ctrl) for 48 h. EVs were isolated from media by differential centrifugation and analyzed by Western blotting for VDAC, cyclophilin D, and GLUD1. (c) Mitochondria (mito.) were purified from cells and EVs. mtDNA was extracted from these preparations and analyzed by long-range PCR. The expected migration position of the mitochondrial nucleoid (∼16 × 10⁶ D) is indicated. (d) EVs were purified by differential centrifugation and incubated in the presence and absence of DNase immobilized to agarose beads. mtDNA and nuclear DNA were determined by qPCR using primers complementary to sequences within the mitochondrial genes (COX1, CYTB, n = 5 independent experiments; or ND1, n = 3 independent experiments) or the nuclear β-globin genes. Values are mean ± SEM. *, P < 0.05; paired t test. (e) Cells were transfected with siRNAs targeting CD63 (siCD63) or a nontargeting siRNA (siNT). Cells were then incubated for 48 h in the absence (Ctrl; NT and siCD63) or presence of 0.3 µM LY95. EVs were purified from the medium by differential centrifugation, and mtDNA content of these was determined using qPCR with primers recognizing the mitochondrial COX1 gene. Values are mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.002; ***, P < 0.001; Mann-Whitney U test. (f) Media collected in the absence (Ctrl) and presence of 0.3 µM LY95 were subjected to differential centrifugation. Differential centrifugation pellets were overlaid with a sucrose density gradient (2–0.4 M sucrose) and centrifuged at 200,000 g overnight. Gradients were eluted, and fractions were collected for analysis by qPCR for the mitochondrial ND1 gene and by Western blotting for CD63. Values represent mean ± SEM, n = 5 independent experiments. *, P < 0.05; Mann-Whitney U test.