Research Paper Volume 13, Issue 1 pp 364—375

Figure 4. Nrf2 binds to a HIF-1A enhancer element in a hypoxia-insensitive manner. (A) HIF-1α mRNA levels were not changed by hypoxia in HepG2 cells. HepG2 cells were incubated under hypoxic condition (5% O₂) for indicated time. mRNA was prepared and RT-qPCR was performed with HIF-1A specific primers. Student’s t-test was performed to evaluate the statistical significance: ns, not significant. (B) HIF-1α mRNA was up-regulated by hypoxia in HepG2 cells. Experiments were performed as in (A). Student’s t-test was performed to evaluate the statistical significance. *P<0.01, **P<0.001. (C) Hypoxia-induced HIF-1α mRNA expression was Nrf2-dependent in HepG2/DDP cells. HepG2/DDP cells were transfected with Nrf2-specific siRNA then incubated under mild hypoxic condition (5% O₂) for indicated time. RT-qPCR was performed as in (A). Student’s t-test: *P<0.01, **P<0.001. (D) Nrf2 hyper-activation increased HIF-1α mRNA expression in HepG2 cells under hypoxia. HepG2 cells were transfected with KEAP1-specific siRNA then incubated with 5% O₂ for indicated time points. RT-qPCR was performed as in (A). Student’s t-test: ***P<0.0001. (E) Diagram showing the conserved Nrf2 binding site at the 5’-end of HIF-1α gene. This site has been shown to be bound by Nrf2 and regulate HIF-1α expression before. (F) Nrf2 binding to HIF-1α enhancer stronger in HepG2/DDP than HepG2 cells and was not sensitive to hypoxia. HepG2 and HepG2/DDP cells were chromatin-immuno-precipitated (ChIP) by using Nrf2-specific antibody. The binding of the conserved site shown in (E) was detected by RT-qPCR. Student’s t-test: ***P<0.0001, ns, not significant. (G) Confirming Nrf2 binding to HIF-1α enhancer by regular PCR. Indicated ChIP samples from (F) were amplified with specific primers to the conserved Nrf2 binding site by regular PCR then subject to DNA electrophoresis.