Fig. 1
From: SIRT1 modulates cell cycle progression by regulating CHK2 acetylation−phosphorylation
SIRT1 binds to and negatively regulates CHK2 phosphorylation. a, b SIRT1 interacts with CHK2 in vivo. HEK293 cell lysates were subjected to immunoprecipitation with control IgG, anti-SIRT1 (a), or anti-CHK2 (b) antibodies. The immunoprecipitates were then blotted with the indicated antibodies. c, d SIRT1 interacts with CHK2 in vitro. Recombinant human SIRT1 (h) or CHK2 (i) was incubated with bacterially expressed GST-CHK2 (h) and GST-SIRT1 (d) for 1 h at 30 °C. e Hydrogen peroxide (H2O2) treatment decreased the binding of CHK2 to SIRT1. Endogenous immunoprecipitation was performed with control IgG or anti-CHK2 antibodies in HEK293 cells treated with 200 μM or without H2O2 for 1 h. Then, the immunoprecipitates were blotted with the indicated antibodies. f SIRT1 knockdown increased the p-CHK2 (CHK2 phosphorylation on Threonine 68 site). Different small interfering RNAs (siRNAs) (#1 and #2) for SIRT1 were transfected into HCT116 cells treated with or without 200 μM H2O2 for 6 h. CHK2 phosphorylation was determined with western blot analysis. g SIRT1+/+ and SIRT1−/− mouse embryonic fibroblasts (MEFs) were treated with or without 200 ng doxorubicin for 12 h. The p-CHK2 level was determined with western blot analysis. h HCT116 cells stably expressing control or Sirt1 short hairpin RNA (shRNA) were irradiated at 5 Gy and released for 1 h. Cell lysates were subjected to western blot analysis. i Catalytic activity of SIRT1 is required for phosphorylation of CHK2. HCT116 cells were transfected into Flag-tagged SIRT1 wild-type (WT) or catalytically inactive mutant H363Y in the absence or presence of 100 μM H2O2. CHK2 phosphorylation on threonine 68 residue (T68) was measured by western blot. j Catalytic activity of SIRT1 inhibition increases CHK2 phosphorylation. HEK293 cells treated with SIRT1 inhibitor EX527 at 0.5 μM for 0, 3, 6, and 9 h were lysed and cell lysates were blotted and measured with the indicated antibodies. k HCT116 cells were treated with or without EX527 at 0.5 μM and Ku55933 at 10 μM for 6 h as indicated, and then cultured in the presence or absence of 100 μM H2O2 for 1 h. Total cell lysates were subjected to western blot analysis. See also Fig. S1
