PINK1 Protects against Oxidative Stress by Phosphorylating Mitochondrial Chaperone TRAP1
Figure 7
TRAP1 Depletion Abolishes the Effects of Wild-Type and Mutant PINK1 on Cell Vulnerability to Oxidative Stress
(A) PC12 cells were transfected with vehicle (control), NT siRNA, or TRAP1-specific siRNAs (TRAP1 siRNA-1 and TRAP1 siRNA-2). The levels of TRAP1 and actin in the cell lysates were analyzed by immunoblotting with anti-TRAP1 and anti-actin antibodies.
(B) PC12 cells treated with TRAP1 siRNA-2 or NT siRNA were either untransfected (UT) or transfected with wild-type (WT) or mutant PINK1 as indicated. Vehicle-treated, non-transfected PC12 cells were used as the control. Cells were exposed to 400 μM H2O2 for 16 h, and the extent of cell survival was assessed by using the MTT assay. Data represent mean ± SEM from three independent experiments. *, p < 0.05; ns, not significant.
(C) PC12 cells co-transfected with pEGFP and vehicle (control) or indicated siRNAs and PINK1 plasmids were treated with 400 μM H2O2 for 16 h, and the extent of apoptosis was determined by morphological analysis of DAPI-stained nuclei. The percentage of transfected cells with apoptotic nuclear morphology was quantified. Data represent mean ± SEM from three independent experiments. *, p < 0.05; ns, not significant.
(D) The levels of cytochrome c (Cyt. c) and actin in the cytosol fractions from cells described in (B) were determined by immunoblotting with anti-cytochrome c and anti-actin antibodies.
(E) The level of cytochrome c released to the cytosol is normalized to the level of actin in each cell sample. Data represent mean ± SEM from three independent experiments. *, p < 0.01; ns, not significant. AU, arbitrary units.
(F) PC12 cells co-transfected with pEGFP and vehicle (control) or indicated siRNAs and PINK1 plasmids were treated with 400 μM H2O2 for 16 h, and the cellular distribution of cytochrome c was detected by immunostaining with anti-cytochrome c antibody. The percentage of transfected cells showing cytochrome c release was quantified. Data represent mean ± SEM from three independent experiments. *, p < 0.05; ns, not significant.