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. 2021 Nov 24;298(1):101448. doi: 10.1016/j.jbc.2021.101448

Figure 4.

Figure 4

Nrf2-KD and p62-KD enhance dopamine-induced cell death.A and B, SH-SY5Y cells were transfected with Nrf2-siRNA (Nrf2-1, 2, or 3) or control (NT) by Lipofectamine RNAiMAX. Cells were treated with 0.4 mM dopamine or DMSO for 4 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. C, SH-SY5Y cells were transfected with Nrf2-siRNA (siNrf2-1, 2 or 3) or control (siNT) by Lipofectamine RNAiMAX. SH-SY5Y cells were treated with 0.2 to 0.4 mM dopamine or DMSO for 12 h. Cells were treated with CCK-8 solution for 1 h. Culture medium was prepared from transfected cells, and the absorbance (485 nm) of culture medium was measured by an absorbance meter (TriStar LB 941). The ratio of absorbance of the cells relative to that of the control siRNA (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗p < 0.0001. NS, not significant. D, SH-SY5Y cells were transfected with p62-siRNA (p62-1 or 3) or control (NT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 4 h or 8 h. Whole cell lysates prepared from transfected cells were characterized by Western blotting using the indicated antibodies. E, SH-SY5Y cells were transfected with p62-siRNA (sip62-1 or 2), USP10-siRNA (siUSP10-1 or 2) and the control (siNT) using Lipofectamine RNAiMAX. Cells were then treated with 0.4 mM dopamine or DMSO for 12 h and with CCK-8 solution for 1 h, and the cell viability was measured with the CCK-8 kit by measuring the absorbance (485 nm) of culture supernatant. The ratio of the absorbance of cells relative to that of the control (siNT) treated with DMSO was presented as the mean and SD from three samples. ∗∗∗∗p < 0.0001.