Fig. 6. ANKRD11 is a p53 target gene. (A) p53 transcriptionally activates a reporter gene driven by an ANKRD11 intronic sequence carrying a p53-RE. HeLa cells were cotransfected with a construct expressing myc-p53 and the reporter constructs pGL3-Basic (left; empty vector), pGL3-Basic-ANKRD11-p53-RE-Luc (middle) or pGL3-Basic-ANKRD11-p53-RE-mut-Luc (right). The relative luciferase activity was determined in cell lysates as described in Fig. 3. Data are represented as mean ± s.e.m. of triplicates. The expression level of the myc-p53 construct compared with endogenous p53 levels in HeLa cells was determined by western blot analysis (inset). (B) ANKRD11 expression correlates with increasing p53 protein levels during the DNA damage response. ANKRD11 expression in either HCT116 or HCT116 (TP53^–/–) cells was determined at time-points 0, 8, 24 and 48 hours post-treatment with mitomycin C. The expression of ANKRD11 in HCT116 (wild-type p53) cells was normalized to that observed in the isogenic HCT116 (TP53^–/–) derivative to monitor only the p53-dependent modulation of ANKRD11 expression during the DNA damage response. Induction of p53 protein levels following treatment with mitomycin C were determined by western blot analysis (inset). (C) HCT116 (p53^–/–) cells were transfected in a 24-well format with the indicated amounts of myc-p53 (A), and the relative ANKRD11 mRNA levels in these treatments were determined using real-time RT-PCR analysis.