Analysis of p53Lys382 acetylation, p53Ser46 phosphorylation, Bax, p300 and Sirt1 protein levels, in HIPK2 knockdown in response to ADR and zinc treatment. (a) RKO cells transfected with pSuper-HIPK2 (siHIPK2) or pSuper control (siRNA C) for 24 h were treated with ADR (2 μg/ml) and ZnCl2 (150 μM) for 16 h. After treatment, equal amounts of total cell extracts were subjected to Western immunoblotting using specific antibodies as indicated. Anti-Hsp70 was used as protein loading control. (b) RKO cell depleted of HIPK2 function by siRNA were treated with ADR and zinc and 24 h and apoptosis was measured by TUNEL assay and shown as percentage of TUNEL positive cells. The results shown are representative of two independent experiments performed in duplicate, ± S.D. (c) Equal amount of nuclear cell extracts of RKO cells depleted of HIPK2 function as in (a) and treated with ADR (2 μg/ml) for 16 h were analysed by Western immunoblotting with anti-p300 antibody. Anti-Hsp70 was used as protein loading control. (d) Equal amount of total cell extracts of RKO-HIPK2-depleted cells as in (a) and treated with ADR (2 μg/ml) for 16 h were analysed by Western immunoblotting with anti-Sirt1 antibody. Anti-tubulin was used as protein loading control.