Fig. 4: B5G1-induced mitophagy is mediated by the PINK1/Parkin pathway.

a After treatment with B5G1 (6 µM) for the indicated times, PINK1 and p-Parkin (Ser65) expression levels were detected by western blotting. β-actin was used as a loading control. b HepG2/ADM cells treated with B5G1 (6 µM) for the indicated times were lysed and then separated into mitochondrial fractions and cytosolic fractions. Parkin translocation was measured by Western blotting. β-actin and VDAC were used as loading controls for the cytosolic and mitochondrial proteins, respectively. c After treatment with B5G1 (6 µM) for 12 h, HepG2/ADM cells were lysed with CO-IP lysis buffer, and the interaction between PINK1 and Parkin was measured by Co-IP assay. The asterisk indicated the band of Parkin. d HepG2/ADM cells were pretreated with NC or PINK1 siRNA, followed by treatment with B5G1 (6 µM) for 12 h. PINK1 expression levels were detected by western blotting. GAPDH was used as a loading control. e HepG2/ADM cells were transfected with NC or PINK1 siRNA for 24 h and then treated with B5G1 (6 µM) for another 24 h. Mitochondrial colocalization with LC3 was observed by a fluorescence microscope. Magnification: ×630; scale bar: 10 μm. f HepG2/ADM cells were transfected with NC or Parkin siRNA for 24 h and then treated with B5G1 (6 µM) for 12 h. Parkin and LC3 expression levels were detected by western blotting. β-actin was used as a loading control. g HepG2/ADM cells were transfected with NC or Parkin siRNA for 24 h and then treated with B5G1 (6 µM) for 24 h. Mitochondrial colocalization with LC3 was detected by immunofluorescence. Magnification: ×630; scale bar: 10 μm