Fig. 4

SIRT7 deacetylates p53 at K320 and K373. a Flag-p53 were overexpressed in Huh7.5 cells, Endogenous SIRT7 or Flag-p53 were immunoprecipitated from cell lysates and the immune complexes were assessed for the presence of p53 or SIRT7 by Western blot. b Deacetylation of p53 by SIRT7 in vitro. The Flag-p53 was purified from HeLa cells treated with or without H₂O₂ (400 μM, 1 h). Purified p53 was incubated in the presence or absence of rSIRT7 with or without NAD⁺. Acetylated and total amount of p53 were assessed by western blot with anti-acetyl-K and p53. c Deacetylation of p53 by SIRT7 in cells. Huh7.5 cells were transfected with siSIRT7 for 72 h and then incubated in the presence of TSA (5 μM) for 2 h. Cells were lysed and cell extracts were subjected to immunoprecipitation with antibody to p53. Acetylated p53 was analyzed by western blot with anti-acetyl-K and total protein was evaluated in whole cell lysates (WCL). d Huh7.5 cells were co-transfected with HA-p53 with Flag-SIRT7 or Flag-SIRT7 HY for 24 h, cells were then incubated with TSA for 2 h. Cells were lysed and cell extracts were subjected to western blot. e Huh7.5 cells were co-transfected wild type Flag-p53 (WT), Flag-p53 K320,373R (2KR), Flag-p53 K320,381,382R (3KR-A), Flag-p53 K120,320,373R (3KR-B), Flag-p53 K372,373,381,382R (4KR), Flag-p53 K120,372,373,381,382R (5KR) with or without HA-SIRT7 for 24 h and then incubated in the presence of TSA for 2 h. Cells were lysed and cell extracts were subjected to immunoprecipitation with magnetic beads to Flag. Acetylated p53 was analyzed by western blot. f Quantification of acetylation levels of p53 mutants in the absence (Control) or presence of SIRT7. Graphs show mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 vs Control, Student’s t-test. g Huh7.5 cells were transfected with siSIRT7 for 48 h and overexpressed 2KR p53, 3KR-A, 3KR-B, Flag-p53 K373,381,382R (3KR-C), 4KR, 5KR for 24 h, acetylated p53 was analyzed as in (d). Data are representative of three independent experiments