FIG 3.

HIF-1α activation of Zp requires p53. (A) Knockout of the TP53 gene eliminates activation of Zp by HIF-1α/β. NOK(p53WT) and NOK(p53KO) cells were cotransfected in parallel with (i) 50 ng of pcDNA3HA-HIF-1α P402A/P564A (expressing a stable variant of HIF-1α) plus 50 ng of pHIF-1β or 100 ng of pcDNA3 as a normalization control and (ii) 200 ng of the indicated luciferase reporter plasmid. Cells were harvested 48 h later, and luciferase activities were determined. Data were normalized to the activities observed with the pcDNA3 control; they represent means ± SEMs of data obtained from assays performed in triplicate on three independent occasions. A pZp-Luc reporter containing the ZIIR element mutation shown in Fig. 6A was used because of poor transfection efficiency of NOK cells; this mutation has no effect on Zp activation by HIF-1α/β (6). (B) Immunoblots showing relative levels of p53 present in the cells used in the experiment shown in panel A.