FIGURE 6.

PINK1 is required for smARF-induced Parkin-dependent mitophagy. A, immunoblot showing PINK1 levels after transfection with PINK1-targeted SMARTpool siRNA (24-h treatment) or with individual siRNAs (PINK1-02, -03, and -04) from the SMARTpool (48-h treatment). B–D, HeLa cells were transfected with NT or PINK1 siRNA (SMARTpool for the recruitment experiment and individual PINK1 siRNAs for the mitophagy experiment). After 48 h, the cells were transduced with eGFP-FLAG-Parkin, and 24 h later, they were transduced with pcDNA, p19WT, p19M1I, and p19ΔNT. After a further 24 h (recruitment experiment) or 48 h (mitophagy experiment), the cells were processed for immunofluorescence using Hsp60 and p19 or p14 antibodies. As a further positive control, cells were treated with 20 μm CCCP for either 3 h (recruitment) or 24 h (mitophagy) at 48 h post-transfection. B, histogram showing the proportion of transfected cells with eGFP-Parkin co-localizing with the mitochondrial marker Hsp60. At least 100 cells were counted per condition per experiment. The PINK1 siRNA condition was compared with NT siRNA for each construct. n = 3. ***, p < 0.001. C, micrographs of NT, PINK1-02 (P-02), and PINK1-04 (P-04) siRNA conditions processed for immunofluorescence after 48 h of p19ΔNT expression. Scale bar, 20 μm. Arrows, smARF- and eGFP-Parkin-transfected cells that have either lost (NT condition) or retained (PINK1-02 and PINK1-04 conditions) their mitochondrial staining. D, histogram showing the proportion of transfected cells that lacked staining for Hsp60 for each of the indicated siRNA conditions. At least 50 cells were counted per condition per experiment. PINK1-02 and PINK-04 siRNA conditions are compared with the NT siRNA condition for each construct and for the 24-h CCCP treatment. n = 3. ***, p < 0.001. Error bars, S.E.