Figure 2.

Knockdown of USP15 enhances mitophagy in HeLa cells, SH-SY5Y cells and primary human fibroblasts. (A) HeLa cells were transfected with empty vector or Parkin cDNA and with siRNAs, as indicated, treated with CCCP (10 µm) for 24 h and immunostained for Parkin and Tom20. The percentage of cells (in the conditions that were not transfected with Parkin) or Parkin-positive cells (in the conditions transfected with Parkin) without mitochondrial staining was quantified (n = 3). *P < 0.001 compared with the CCCP-treated condition transfected with control (Ctrl.) siRNA and Parkin cDNA. (B and C) SH-SY5Y cells were transfected with the indicated siRNAs, treated with CCCP (25 µm) for 24 h and immunostained for Hsp60. (C) Quantification of the percentage cells without mitochondrial staining (n = 3). *P < 0.005 compared with the CCCP-treated control siRNA condition. (D–G) Fibroblasts from healthy control 1 were treated with DMSO, CCCP (10 µm), CCCP + bafilomycin (Baf., 100 nm), valinomycin (Val., 1 µm) or valinomycin + bafilomycin for 24 or 48 h, as indicated, followed by anti-Hsp60 immunostaining (D and E) or western blot (F and G). (E) Quantification of the percentage cells without detectable Hsp60 immunoreactivity (n = 3). *P < 0.001 compared with the 48 h DMSO condition. (G) Quantification of the Hsp60/β-Actin ratio on western blot, normalized to this ratio in the DMSO condition (n = 7). *P < 0.005 compared with the 48 h DMSO condition. (H–K) Fibroblasts from control 1 were transfected with the indicated siRNAs and treated with DMSO, CCCP or valinomycin for 24 h, followed by anti-Hsp60 immunostaining (H, I) or western blot (J and K). (I) Quantification of the percentage cells without detectable Hsp60 immunoreactivity (n = 3). * P ≤ 0.01 compared with the CCCP-treated control siRNA condition. (K) Quantification of the Hsp60/β-Actin ratio on western blot, normalized to this ratio in the DMSO condition (n = 5). * P < 0.05 compared with the DMSO condition.