Fig. 7

miR-27a/b prevent Parkin translocation to mitochondria upon mitochondrial damage. a, b 48 h post-transfection, HeLa cells were incubated with 10 μM CCCP for 2 h. After isolating the mitochondria-enriched fractions, Parkin translocation and LC3-II were assessed by Western blot. Each protein level was normalized to corresponding mitochondrial loading control (SOD2) level and quantified as a percentage of control (n = 3, two-way ANOVA). Purity of mitochondrial fraction was assessed by monitoring p38 cytosolic and VDAC1 mitochondrial markers. PN; post-nuclear, C; cytoplasm, M; mitochondrial (A). c, d 48 h post-transfection, HeLa cells stably expressing GFP-Parkin were visualized by GFP, a mitochondrial marker (TOM20) antibody, and a nuclear dye (Hoechst) as indicated. Scale bars correspond to 10 μm. Parkin translocation was quantified as the ratio of cytoplasmic to nuclear GFP signal and the resulting ratios were normalized to control. Data were collected from 4 independent replicates (n > 1000 cells) and are shown as a percentage of control. Values are mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001)