Fig. 2.

Knockdown of Pink1 or Park2 suppresses LPS-induced mitophagy in RPTC cells. (A-C) RPTC cells were subjected to 100 μg/ml LPS treatment for indicated time to collect whole cell lysate for immunoblot analysis of PINK1, PARK2, and GAPDH. (A) Representative blots. (B, C) Densitometry analysis of PINK1 and PARK2. (D, E) Silencing of Pink1 or Park2 expression by siRNA in RPTC cells. RPTC cells were transfected with negative control siRNA (NC siRNA), Pink1 siRNA and Park2 siRNA for 48 h to collect whole cell lysate for immunoblot analysis of PINK1, PARK2 and GAPDH. (F, G) Preservation of TOM20 and TIM23 by silencing of Pink1 and Park2 during LPS treatment of RPTC cells. RPTC cells were transfected with Pink1 siRNA and Park2 siRNA alone or NC siRNA. At 48 h after transfection, the cells were subjected to LPS treatment to collect whole cell lysate for immunoblot analysis of TOM2O, TIM23, and GAPDH. (H, I) Inhibition of LPS-induced mitophagosome formation by silencing Pink1 or Park2. RPTC cells were first transfected NC siRNA, Pink1 siRNA and Park2 siRNA, and 24 h later these cells were transfected with COX8-EGFP-mCherry. After 8 h, the cells were subjected to LPS treatment. Finally, the cells were stained with DAPI (blue) and fixed for confocal microscopy. (H) Representative images. (I) Quantification of the cells with red-only puncta. Data in I are expressed as mean ± SD. n = 3. *P < 0.001 vs. the control group, #P < 0.05 vs. NC siRNA-LPS group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)