Figure 6. Phosphorylation at Thr344 inactivates SIRT1 deacetylase activity.

A) Pre-acetylated p53 was incubated with wild-type or mutants of His-SIRT1 in the presence or absence of NAD⁺, and the level of p53 acetylation was detected by acetylation-specific anti-p53 antibody. B) HepG2 cells were transfected with DNAs encoding wild type or mutant Flag-SIRT1, and co-treated with 100 μM etoposide overnight. The level of p53 acetylation was detected and quantified. Results are mean ± SD; * P<0.05. C) His-tagged mutants of SIRT1 were pulled down with GST, GST-p53 or GST-acetyl-p53. WT – wild type; TA – T344A; TE – T344E. D) 0.3 or 0.9 μg of DNA encoding SIRT1 mutants was transfected into HepG2 cells together with a p53-luciferase reporter, and luciferase acitivity measured. Results are mean ± SD; *, P<0.05; **, P<0.01. E) HepG2 cells were transfected with DNAs encoding wild type or mutant SIRT1 and treated with 100 μM etoposide and 0.5 μM TSA for 6 hours. The transcript level of p21^CIP and Bax were measured using RT-qPCR. results are mean ± SD; *, P<0.001. F) SMMC-7721 cells were transfected with DNAs encoding GFP-SIRT1 and treated with 100 μM etoposide for 24 hours. Cells were stained with Annexin V- PE and 7-AAD. Only GFP-positive cells were gated for analysis. (G) HepG2 cells were transfected with DNAs encoding SIRT1 for colony formation assays. Results are mean ± SD; *, P<0.01. H) A diagram summarizes the role of AMPK in stabilizing p53. P: phosphate, Ac: acetyl.