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. 2019 Aug 26;16(4):724–734. doi: 10.1080/15548627.2019.1656957

Figure 3.

Figure 3.

USP33 depletion promotes PRKN recruitment to depolarized mitochondria. U2OS-GFP-PRKN cells were transfected with control siRNA (siCon) or USP33 siRNA1 + 2 (siUSP33) for 48 h and then treated with 20 μM CCCP. The cells were collected at indicated time-points for immunostaining with anti-TOMM20. (A) The appearance of GFP-PRKN puncta superposed on the mitochondrial TOMM20 was visualized. Scale bar: 25 μm. (B) Knockdown of USP33 validated by western blotting. (C) Percentage of cells with GFP-PRKN puncta colocalized with TOMM20 was quantified in siControl and USP33-depleted cells at 5-min interval. Over 150 cells were examined at each time-point, and data represents mean ± SD from three independent experiments. One-way ANOVA was used for statistical analysis. *P < 0.05. (D) Interaction between PRKN and USP33 in mitochondrial fraction of HEK293 cells with or without CCCP (20 μM) treatment for 2 h.