Figure 2. PHLDB3 is a direct transcriptional target gene of p53.

(a,b) The effect of p53 overexpression on PHLDB3 mRNA (a) and protein levels (b). H1299 cells were transfected with p53 and harvested 36 h post-transfection. The relative PHLDB3 mRNA level was quantified by RT-qPCR. Data was corrected for GAPDH and represent mean±s.e.m. of triplicate experiments. *P<0.01 by two-tailed t-test (a). The PHLDB3, p53 and p21 levels were determined by immunoblotting with corresponding antibodies (b). (c) Two potential p53 responsive elements (RE1 and RE2) were identified in the human PHLDB3 promoter region using computer software (p53MH algorithm). The CATG to TATA mutation of RE1 was generated by site-directed mutagenesis. (d) p53 induces the activity of luciferase, whose expression was driven by the RE1, but not RE2 or mutated RE1, in the PHLDB3 promoter. Data represent mean±s.e.m. of triplicate experiments.*P<0.01 by two-tailed t-test. (e) p53 binds to the PHLDB3 promoter in cells. HCT116^p53+/+ cells were treated with 1 μM DOX for 16 h, and ChIP assays were performed with the p53 antibody or control IgG. The promoter regions of indicated genes were analysed by RT-qPCR. *P<0.01 by t-test.