Figure 5

Nicotinamide reconstitutes p53Lys382 acetylation and apoptotic transcriptional activity. (a) RKO cells stable depleted of HIPK2 function were treated with ADR (2 μg/ml) and zinc (150 μM) for 16 h in the presence of nicotinamide (Nic) or TSA. Equal amounts of nuclear extracts were analysed by Western immunoblotting of Lys382, Ser46, total p53, p21 and Bax. Hsp70 represents protein loading control. (b) RKO cells stable depleted of HIPK2 function were transfected with p53AIP1-luc reporter for 24 h and treated with ADR and zinc for 16 h in the presence of Nic or TSA before luciferase activity was assayed. Results, normalized to β-galactosidase activity, are representative of three independent experiments performed in duplicate ± S.D. * P < 0.01. (c) H1299 cells stable depleted of HIPK2 function were co-transfected with low amount of wtp53 expression vector and p21-luc, GADD45-luc, p53AIP1-luc, and Noxa-luc reporters and 24 h after transfection treated with ADR and zinc for 16 h in the presence of Nic or TSA before luciferase activity was assayed. Results, normalized to β-galactosidase activity, are representative of three independent experiments performed in duplicate ± S.D. * P < 0.01.