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. 2020 May 4;130(6):2953–2965. doi: 10.1172/JCI123159

Figure 3. SIRT2 regulates TC-NER–mediated DNA cross-link repair.

Figure 3

TC-NER efficiency was analyzed using a TC-NER reporter assay. Luciferase expression and activity from a cisplatin-induced cross-linked CMV–firefly luciferase plasmid was calculated as a percentage of the expression/activity from a CMV–firefly luciferase control plasmid. (A) XPA-mutated cells were defective in NER-mediated repair of the cross-linked luciferase plasmid compared with XPA-complemented (C-XPA) cells (n = 5). (B) NER-mediated repair of cross-links was diminished in Sirt2-KO neuronally differentiated 50B11 and PC12 cells. Reexpression of WT-SIRT2, but not the enzymatically inactive mutant, HY-SIRT2, in KO cells restored NER efficiency (n = 3). Western blots (left inset) show rat SIRT2 (rSIRT2) and human SIRT2 (hSIRT2) expression in these cells. Vinculin was the protein loading control. (C) Dot blot for level of DNA-platinum adduct measurement in neuronally differentiated 50B11 cells following cisplatin or saline administration (n = 3). (D) Quantification of DNA dot blot (n = 2). Sirt2 KO resulted in an increase in DNA-platinum adduct formation following cisplatin treatment. Reexpression of WT-SIRT2 restored DNA-platinum adduct repair. Statistical significance was assessed using 2-tailed Student’s t test with Welch’s correction (A), 1-way ANOVA with Dunnett’s correction (B), or 2-way ANOVA with Bonferroni’s correction (D). **P < 0.01; ***P < 0.001.