HREs can be used to induce p53 in response to HIF-1α levels. (A) Schematic representation of the inducible p53 expression constructs used in the analysis. Human p53 is shown; PP is the polyproline region. (B) 5× HRE mouse p53 constructs were transfected into H1299 cells. The cells were then exposed to 2% O2 for a period of 16 h. Western blots were carried out for HIF-1α, p53, and, as a loading control, GAPDH. (C) H1299 cells transfected with 5× HRE mouse p53 constructs were exposed to hypoxia (0.02% O2) for 8 h and fixed under hypoxic conditions. The cells were stained for total p53 and α-tubulin. The localizations of 5× HRE mp53wt, 5× HRE mp5325,26, and 5× HRE mp53P/A are shown. (D) MEFs (p53−/−) were transfected with 5× HRE mp53wt, 5× HRE mp5325,26, and 5× HRE mp5325,26,53,54; exposed to 2% O2; processed for ChIP as described for Fig. ; and analyzed by PCR using primers specific for the Ankyrin-like repeat protein, Apaf-1, and p21 promoters. Inputs and samples were titrated to determine the quantity of p53 bound to a given promoter. Quantities of binding are shown below the corresponding lanes on a representative gel.