Figure 4.

Effects of TEPA on HIF-1α binding to HRE sites of BNIP3 or IGF2. A and B, effect of TEPA on the binding of HIF-1α to the BNIP3–HRE1 detected by ChIP assay under CoCl₂ (A) or hypoxia treatment (B). C and D, luciferase reporter assay detection of the effect of TEPA on the transcriptional function mediated by BNIP3–HRE1 under CoCl₂ (C) or hypoxia treatment (D). E, effect of TEPA on mRNA expression of BNIP3 in cells treated with CoCl₂ detected by qRT-PCR. F and G, effect of TEPA on the binding of HIF-1α to the IGF2–HRE2 detected by ChIP assay under CoCl₂ (F) or hypoxia treatment (G). H and I, luciferase reporter assay detection of the effect of TEPA on the transcriptional function mediated by IGF2–HRE2 under CoCl₂ (H) or hypoxia treatment (I). J, effect of TEPA on mRNA expression of IGF2 detected by qRT-PCR under CoCl₂ treatment. A, C, E, F, H, and J, cells treatments were as described for Fig. 3F. B, D, G and I, HUVECs were cultured under hypoxic condition (1% O₂) for 8 h only (Hypoxia), or treated with 50 μm TEPA for 32 h only (TEPA) or cultured cells under hypoxic condition for 8 h, with a pretreatment of 50 μm TEPA, for a total of 32 h (including the final 8 h for hypoxia treatment) (Hypoxia+TEPA); an untreated group served as control (Control). The plasmid used in the luciferase reporter assay (C, D, H, and I) was PGL3-promoter containing WT BNIP3–HRE1 or WT IGF2–HRE2. At least three independent experiments were carried out. *, significantly different from control group (p < 0.05); #, significantly different from Co or hypoxia group (line 2) (p < 0.05) detected by two-way ANOVA.