Figure 2.

USP10-KD augments dopamine-induced ROS production and ROS-dependent cell death.A and B, SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-2) or control (siNT) using Lipofectamine RNAiMAX. Cells were treated with 5 μM CM-H2DCFDA (Green), an ROS-sensitive fluorescence dye, for 30 min. Transfected cells were then treated with 0.4 mM dopamine or DMSO for 6 h. The fluorescence intensity was measured. The ratio of fluorescence intensity of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗p < 0.05; ∗∗∗∗p < 0.0001. Scale bar is 10 μm. Bright-field observations of cells were also presented. C, SH-SY5Y cells were transfected with USP10-siRNA (siUSP10-1 or 2) or control (siNT) using Lipofectamine RNAiMAX. Cells were pretreated with 500 μM N-acetyl cysteine 30 min before dopamine treatment and further treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h. Cell viability was evaluated by measuring the absorbance (485 nm) of culture medium with an absorbance meter (TriStar LB 941). The ratio of absorbance obtained from cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗p < 0.0001. D, USP10-KD (siUSP10-2) and control (siNT) cells were treated with 50 to 200 μM H₂O₂ for 12 h. Cell viability was measured with the CCK-8 kit. The absorbance obtained from cells was normalized to that of the control (siNT) treated with DMSO, and the ratio was presented as the mean and SD from three samples. ∗∗p < 0.01; ∗∗∗∗p < 0.0001. NS, not significant; USP10, ubiquitin-specific protease 10.