Figure 6. SIRT2 protection of neuronally differentiated cells from cisplatin cytotoxicity is dependent on NER function.

(A) Western blot showing XPB and SIRT2 expression in neuronally differentiated 50B11 cells with varying Sirt2 gene modifications with or without spironolactone (SP) treatment. The lanes were from the same gel but were not contiguous. Blots represent 1 of 3 replicates. (B) NER efficiency was significantly decreased by Sirt2 KO or treatment of 50B11 cells with 10 μM SP, which can increase the degradation of the key NER protein, XPB, and thereby inhibit NER. Sirt2 KO led to significantly lower NER than SP. Reexpression of WT-SIRT2 rescued NER only in the absence of SP-mediated interruption of NER function. n = 5 or 3. (C) SP pretreatment increased cisplatin-induced cytotoxicity in 50B11 cells. SP had no further effect on cisplatin-induced cell killing in Sirt2-KO and NER-deficient 50B11 cells. WT and Sirt2-KO cells were rescued with WT-SIRT2 to rule out off-target effects and showed no difference in survival. The viability of 50B11 cells after cisplatin treatment at various doses was measured with trypan blue staining. Statistical significance was assessed using 2-way ANOVA with Bonferroni’s correction (B and C). **P < 0.01; ***P < 0.001.