Figure 4: Cytosolic p53 interrupts the mitochondrial translocation of Parkin.

(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments. (b) These samples were subjected to immunoprecipitation with the anti-VDAC antibody to evaluate ubiquitination. Representative immunoblots are shown from three independent experiments. (c,d) Representative images of YFP-Parkin overexpressing p53^−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8 (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm. (e–g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300–400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P<0.05, compared with the corresponding control (two-tailed unpaired Student’s t-test).