Figure 3. PINK1 controls production of... | Rockefeller University Press

Figure 3.

$PINK1 controls production of mtDNA-containing EVs. (a and b) MDA-MB-231 cells were transfected with an siRNA targeting PINK1 (siPINK1) or a nontargeting control siRNA (siNT). EVs released from transfected cells were analyzed using nanoparticle tracking and Western blotting for CD63 or CD9 as in Fig. 1 a. Values represent the mean, and dotted line and error bars represent the SEM; n = 5 independent experiments. *, P < 0.05; t test with Welch’s correction (a). Differential centrifugation pellets were further fractionated using sucrose density gradient centrifugation and analyzed by qPCR for mtDNA and by Western blotting for CD63 as in Fig. 2 f (b). Values represent mean ± SEM; n = 3 independent experiments. *, P < 0.05; Mann-Whitney U test. (c and d) Cells were transfected with an siRNA targeting PINK1 (siPINK1) or nontargeting control siRNA (siNT) in combination with rescue vectors for WT or kinase-dead (KD) PINK1-YFP. Cells were treated with CCCP as indicated, lysed, and analyzed by Western blotting for PINK1 or phosphorylated Ub with actin as a loading control (c). EVs were collected over a 48-h period and analyzed using nanoparticle tracking as in Fig. 1 a (d). Values represent the mean of four independent experiments; dotted line and error bars are SEM. *, P < 0.01; ****, P < 0.0001; one-way ANOVA with Dunn’s multiple comparison test.$

PINK1 controls production of mtDNA-containing EVs. (a and b) MDA-MB-231 cells were transfected with an siRNA targeting PINK1 (siPINK1) or a nontargeting control siRNA (siNT). EVs released from transfected cells were analyzed using nanoparticle tracking and Western blotting for CD63 or CD9 as in Fig. 1 a. Values represent the mean, and dotted line and error bars represent the SEM; n = 5 independent experiments. *, P < 0.05; t test with Welch’s correction (a). Differential centrifugation pellets were further fractionated using sucrose density gradient centrifugation and analyzed by qPCR for mtDNA and by Western blotting for CD63 as in Fig. 2 f (b). Values represent mean ± SEM; n = 3 independent experiments. *, P < 0.05; Mann-Whitney U test. (c and d) Cells were transfected with an siRNA targeting PINK1 (siPINK1) or nontargeting control siRNA (siNT) in combination with rescue vectors for WT or kinase-dead (KD) PINK1-YFP. Cells were treated with CCCP as indicated, lysed, and analyzed by Western blotting for PINK1 or phosphorylated Ub with actin as a loading control (c). EVs were collected over a 48-h period and analyzed using nanoparticle tracking as in Fig. 1 a (d). Values represent the mean of four independent experiments; dotted line and error bars are SEM. *, P < 0.01; ****, P < 0.0001; one-way ANOVA with Dunn’s multiple comparison test.