Figure 3

Ectopic expression of p300 in HIPK2 knockdown rescues the p53Lys382 apoptotic transcriptional activity. (a) RKO cells stable-transfected with p53AIP1-luc reporter were transfected with pSuper-HIPK2 (siHIPK2) or control pSuper (siRNA) vectors for 24 h and then treated with ADR (2 μg/ml) and zinc (150 μM) for 16 h before luciferase activity was assayed. Results are representative of three independent experiments performed in duplicate ± S.D. * P < 0.01. (b) H1299 cells were co-transfected with p53AIP1-luc reporter and HIPK2-Flag together with wtp53, Q373, R382, or A46Q373 mutants. Luciferase activity was assayed 36 h thereafter. (c) RKO cells stable depleted of HIPK2 function were transfected with p300-Flag expression vector and treated with ADR for 16 h. Equal amount of total cell extracts were immunoprecipitated with conformation-specific PAb240 (for p53 mutant, unfolded conformation) antibody and immunoblotted (IB) with anti-p53 (FL393) antibody (upper panel). Endogenous p300 (arrow) and ectopic p300-Flag protein levels were detected by immunoblotting (IB) equal amount of total cell extracts with anti-p300 antibody (lower panel), endogenous p53 levels were also detected. Anti-tubulin was used as protein loading control (lower panel). (d) RKO cells stable depleted of HIPK2 function were transfected with p300-Flag expression vector and treated with ADR and zinc for 16 h. Equal amount of total cell extracts were immunoblotted with anti-phospho-Ser46, anti-acetyl-Lys382, anti-p53, and anti-PARP (arrows indicate the uncleaved and cleaved forms) antibodies. (e) RKO cells stable depleted of HIPK2 function were co-transfected with the indicated reporters along with p300 expression vectors for 24 h and then treated with ADR for 16 h 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.