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. 2019 Apr 26;12:108. doi: 10.3389/fnmol.2019.00108

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Copyright © 2019 Zhang, Hong, Cao, Yin, Shi and Ying.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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SIRT2 silencing prevented NAD⁺-induced increases in the glutathione levels, GSH/GSSG ratio, GCL level, and Nrf2 level of PC12 cells. (A) SIRT2 siRNA decreased the protein levels of SIRT2 in PC12 cells. (B) SIRT2 silencing prevented the NAD⁺-induced increase in the GSH level of the cells (effect of NAD⁺ treatment, F_(1,44) = 105.2, p < 0.0001; effect of SIRT2 siRNA, F_(1,44) = 16.01, p = 0.0002; effect of SIRT2 siRNA * NAD⁺ treatment, F_(1,44) = 50.4, p < 0.0001). (C) Effects of SIRT2 siRNA and NAD⁺ on GSSG levels (effect of NAD⁺ treatment, F_(1,44) = 1.544, p = 0.2206; effect of SIRT2 siRNA, F_(1,44) = 0.0108, p = 9177; effect of SIRT2 siRNA * NAD⁺ treatment, F_(1,44) = 3.279, p = 0.077). (D) SIRT2 silencing prevented the NAD⁺-induced increase in the GSH/GSSG ratio of the cells (effect of NAD⁺ treatment, F_(1,44) = 12.21, p = 0.0011; effect of SIRT2 siRNA, F_(1,44) = 4.623, p = 0.0371; effect of SIRT2 siRNA * NAD⁺ treatment, F_(1,44) = 3.336, p = 0.0746). (E) SIRT2 silencing prevented the NAD⁺-induced increase in the total glutathione level of the cells. (effect of NAD⁺ treatment, F_(1,44) = 123.6, p < 0.0001; effect of SIRT2 siRNA, F_(1,44) = 13.73, p = 0.0006; effect of SIRT2 siRNA * NAD⁺ treatment, F_(1,44) = 56.69, p < 0.0001). (F) SIRT2 silencing prevented the NAD⁺-induced increase in the GCLc mRNA level of the cells (effect of NAD⁺ treatment, F_(1,44) = 5.452, p = 0.0242; effect of SIRT2 siRNA, F_(1,44) = 8.229, p = 0.0063; effect of SIRT2 siRNA * NAD⁺ treatment, F_(1,44) = 10.69, p = 0.0021). (G) SIRT2 silencing prevented the NAD⁺-induced increase in the GCLm mRNA level of the cells (effect of NAD⁺ treatment, F_(1,44) = 32.68, p < 0.0001; effect of SIRT2 siRNA, F_(1,44) = 10.09, p = 0.0027; effect of SIRT2 siRNA * NAD⁺ treatment, F_(1,44) = 4.664, p = 0.0363). (H) SIRT2 silencing prevented the NAD⁺-induced increase in the Nrf2 mRNA level of the cells (effect of NAD⁺ treatment, F_(1,44) = 305, p < 0.0001; effect of SIRT2 siRNA, F_(1,44) = 178.7, p < 0.0001; effect of SIRT2 siRNA * NAD⁺ treatment, F_(1,44) = 178.7, p < 0.0001). The cells were treated with SIRT2 siRNA for 6 h. After the media was replaced with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, the cells were treated with NAD⁺. RT-PCR assays were conducted at 6 h after the NAD⁺ treatment, while glutathione assays were conducted at 24 h after the NAD⁺ treatment. The data were pooled from four independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.