Tollip suppress CCCP-induced mitophagy. (A) HeLa cells overexpressing wild-type parkin were transfected with wild-type Tollip, scrambled siRNA (control siRNA), and/or PINK1-siRNA for 24 h and then treated with either DMSO (control) or 20 μM CCCP for an additional 24 h. Scale bar = 10 μm. Mitochondrial loss in each cell type was quantified (bottom graph, **P ≤ 0.01). (B) PINK1+/+ and PINK1−/− MEFs were mock-transfected or transfected for 24 h with Xpress-Tollip and treated with DMSO or 20 μM CCCP for 4 h. Mitochondrial membrane potential of each sample was then evaluated using JC-1 staining. Scale bar = 10 μm. (C) PINK1+/+ and PINK1−/− MEFs were mock-transfected or transfected with Xpress-Tollip for 24 h, followed by treatment with DMSO or 20 μM CCCP for 72 h. The level of ATP in each sample was then measured using an ATP Determination Kit. (D) SH-SY5Y cells were either mock-transfected or transfected with Xpress-Tollip for 24 h and then treated with 10 μM CCCP for 4 h.
