SIRT2 regulates cisplatin-induced tubular apoptosis and necroptosis. (A and B) Apoptosis was assessed by the terminal deoxynucleotidyl TUNEL assay and the number of apoptotic cells, as defined by chromatin condensation or nuclear fragmentation (apoptotic bodies), was counted. Scale bar = 100 µm. Kidneys in WT, Sirt2 KO and Sirt2 TG mice were harvested 3 days after cisplatin (Cis) or CB injection. TUNEL-positive cells were counted in 10 randomly, non-overlapping fields (×400 magnification) per kidney (n = 8, each group). The number of TUNEL-positive cells was significantly decreased in Sirt2 KO mice kidney and increased in Sirt2 TG mice kidney compared with WT mice. (C and D) Cleaved caspase-3 expression was evaluated by immunofluorescence (C) and Western blot analysis (D) using anti-caspase-3 (detecting cleaved forms) antibody. Kidneys in WT (n = 5), Sirt2 KO (n = 5) and Sirt2 TG (n = 5) mice were harvested 3 days after cisplatin administration. Densitometric analysis for cleaved caspase-3 is presented as the relative ratio of each cleaved caspase-3 to GAPDH. (E) Flow cytometry analyses of Annexin-V in small interfering RNA-targeting SIRT2 (SIRT2–siRNA) or control-siRNA (Cont-siRNA)-transfected MPT cells at 24 h after cisplatin (Cis; 20 mg/mL) treatment (n = 4, each group). (F) Western blot analysis of RIP-1. Kidneys in WT (n = 5), Sirt2 TG (n = 5) and Sirt2 KO (n = 5) mice were harvested 3 days after cisplatin administration. Data are expressed as mean ± SD. **P < 0.01 versus WT + CB; ⁺P < 0.01 versus WT + Cis; ^#P < 0.05 versus WT + Cis; ^$$P < 0.05 versus CB + Cont-siRNA; ^{^}P < 0.05 versus Cis + Cont-siRNA.