Figure 4.

Cooperation of HIF-1α⁴¹⁷ and ARNT in HRE-reporter gene transcription. (A) A luciferase reporter plasmid containing the Epo enhancer was co-transfected into HEK293 cells with various doses of pHIF417. The reporter activity was measured in lysates from cells cultured under normoxic conditions. Western blotting in the right panel demonstrated that HIF417 was expressed in a gene dose-dependent manner and endogenous HIF-1α levels were not affected by HIF417 expression. (B) Various doses of pARNT (or pARNT603) and pHIF417 were co-transfected into cells using the EPO reporter. Western blotting in the right panel demonstrated the expression of ARNT and ARNT603 proteins. (C) Various doses of pHIF417 and pARNT (or pARNT603) were co-transfected into cells using the EPO reporter. (D) The mutated Epo reporter lacking the HRE core sequence was co-transfected into cells with pARNT and pHIF417. Bars represent the mean and SD of six or more experiments. Statistical differences were compared using the unpaired two-tailed Student's t-test. ^*: P < 0.05 versus the control group transfected with only the reporter plasmid. #: P < 0.05 versus the group co-transfected with both pHIF417 and pARNT. (E) Total RNAs were isolated from untransfected or transfected Hep3B cells subjected to normoxia (lanes 1–4) or 16 h hypoxia (lane 5), and erythropoietin (EPO) mRNA was analyzed by semi-quantitative RT–PCR. Cells were transfected with 0.5 μg of pARNT and various doses of pHIF417. β-actin mRNA was analyzed as a loading control.