Fig. 7

NIPSNAP1 increases the antioxidant activity of SOD2 through SIRT3-mediated deacetylation. A, B HCT116 (left) and HepG2 (right) cells transduced with sh-Ctrl or two independent-shRNAs targeting (sh-NIPSNAP1-1 and -2, respectively) were subjected to Western blotting to measure NIPSNAP1 and SOD2 levels (A) or manganese superoxide dismutase (MnSOD) activity (B), respectively. C, D HCT116 cells transduced with sh-Ctrl or two independent-shRNAs targeting (sh-NIPSNAP1-1 and -2, respectively) (C) or with pCDH or the pCDH-NIPSNAP1 overexpression vector (D) were co-transfected with 3*Flag-SOD2. Thereafter, cell lysates were subjected to immunoprecipitation (IP) with anti-Flag antibodies followed by Western blot analysis against Flag, and generalized antibodies recognizing acetylated lysines, phosphorylated tyrosine (p-Tyr) or phosphorylated tyrosine/serine residues (p-Ser/Thr), respectively. Input samples were analyzed in parallel to confirm knockdown and overexpression efficiency, respectively. E HCT116 cells with knockdown (left) or overexpression of NIPSNAP1 (right) as per C, D were analyzed by Western blot to measure the levels of K68 and K122 acetylated SOD2. F HCT116 cells transduced with pCDH or pCDH-NIPSNAP1 were co-transfected with 3*Flag-SOD2 before culture with or without NAM (nicotinamide, 10 mM) or TSA (Trichostatin A, 1 μM). Thereafter, cell lysates were subjected to immunoprecipitation (IP) with anti-Flag antibodies followed by Western blot analysis against Flag, and generalized antibodies recognizing acetylated lysine. G HCT116 cells transduced with sh-SIRT2 or sh-SIRT3 or sh-SIRT4 or sh-SIRT5 or sh-SIRT7 in combination with pCDH-NIPSNAP1 were subjected to Western blot analysis against NIPSNAP1, SIRT isoforms or P27, respectively. H, I HCT116 cells transduced with sh-SIRT3 in combination with pCDH-NIPSNAP1 were subjected to Western blot analysis against NIPSNAP1, SIRT3, K68 and K122 acetylated and total SOD2, respectively (H) or to flow cytometric analysis of ROS levels (I). J Immunoprecipitation (IP) analyses undertaken in HCT116 cells examining interactions between endogenous NIPSNAP1 and SIRT3. K, L Immunoprecipitation (IP) analyses undertaken in HCT116 cells examining the effect of NIPSNAP1 knockdown (K) and overexpression (L) on interactions between SIRT3 and SOD2. HCT116 cells as per C and D were subjected to IP with anti-Flag antibodies followed by Western blot analysis to detect SOD2-3*Flag (anti-Flag) and SIRT3. Input samples were analyzed in parallel to confirm the effects of NIPSNAP1 knockdown and overexpression, respectively, and equal input levels of SIRT3. B Is mean ± SD; n = 3 independent experiments, two-tailed Student’s t test, Images in A and C–L represents three independent experiments (*P < 0.05; **P < 0.01, ***p < 0.001)