Fig. 3
From: TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage
TSPYL2 regulates p53 acetylation by inhibiting SIRT1 and promoting p300 activity. a p53 was immunoprecipitated from etoposide treated U2OS cells and incubated in vitro with cell extracts obtained from control or TSPYL2-depleted cells, also silenced for p53, in presence or absence of the SIRT1 inhibitor nicotinamide (NAM [25]). p53 acetylation/total p53 ratio was analyzed by western blot and normalized to the value of NAM-treated siLUC-transfected cells. Results of three independent experiments were reported in the chart. Error bars represent s.d. b U2OS cells were transfected with control, TSPYL2 and SIRT1 siRNAs in different combinations. After 48 h transfection, cells were treated with MG132 followed by etoposide (20 µM, 3 h) and p53 acetylation was analyzed by western blot. c p300 was immunoprecipitated from control or TSPYL2-depleted cells and its activity tested against recombinant GST-p53 in presence or absence of acetyl-coA. p53 acetylation/total p53 ratio was analyzed by western blot and normalized to the value of siLUC-transfected cells supplemented with acetyl-coA. Results of three independent experiments were reported in the chart. Error bars represent s.d. d U2OS cells were transfected with control or TSPYL2 siRNAs and MOCK or p300 expression vectors in different combinations. After 48 h, cells were treated with MG132 followed by etoposide and p53 acetylation was analyzed by western blot. e U2OS cells were transfected with control, TSPYL2, SIRT1, and p300 siRNAs in different combinations. After 48 h of transfection, cells were treated with MG132 followed by etoposide (20 µM, 3 h) and p53 acetylation was analyzed by western blot. The fold induction of acetylated p53 relative to total p53 is indicated in each western blot a–c. f p300 was immunoprecipitated from SIRT1-WT or SIRT1-KO cells and its activity tested against recombinant GST-p53 in presence or absence of acetyl-coA. p53 acetylation/total p53 ratio was analyzed by western blot and normalized to the value of SIRT1-WT cells. Results of three independent experiments were reported in the chart. Error bars represent s.d.
