FIG. 3.

Effect of heregulin stimulation on HIF-1 transcriptional activity. Transfected cells were serum starved and exposed to no growth factor (−), 10% FBS, heregulin (HRG) or FBS and HRG for 24 h, under either nonhypoxic (20% O₂) (top panels) or hypoxic (1% O₂) (bottom panels) culture conditions, prior to preparation of cell lysates for dual luciferase assays. (A) Analysis of HIF-1-mediated reporter gene transcription. MCF-7 cells were cotransfected with pSV-Renilla and p2.1 or p2.4, which contain a wild-type and mutated HRE, respectively. The ratio of firefly to Renilla luciferase expression was determined and normalized to the value obtained from nonhypoxic cells transfected with p2.1 (Relative Expression). (B) Analysis of HIF-1α transactivation domain function. MCF-7 cells were transfected with: pSV-Renilla; pG5E1bLuc, which contains five copies of a GAL4 DNA-binding site upstream of the Elb promoter and firefly luciferase coding sequences; and either pGal0 or pGalA, which encodes the GAL4 DNA-binding domain either alone or fused to HIF-1α amino acids 531 to 826, respectively. The ratio of firefly to Renilla luciferase expression was determined and normalized to the value obtained from nonhypoxic cells transfected with pGal0 (Relative Expression).