Research Paper Volume 13, Issue 1 pp 364—375

Figure 2. Nrf2 hyper-activation is required for sustained HIF-1α induction in HepG2/DDP cells. (A) Up-regulation of Nrf2 by KEAP1 knockdown. HepG2 and HepG2/DDP cells were transfected with siRNA specific to Nrf2 or KEAP1. Protein levels of Nrf2, KEAP1 and actin control were examined by Western blot. (B) Quantification of 3 experiments in (A). Student’s t-test was performed to evaluate the statistical significance. *P<0.01, **P<0.001. (C) Nrf2 knockdown blocked the sustained induction of HIF-1α in HepG2/DDP cells by hypoxia. Cells were transfected with Nrf2-specific siRNA for 24 hours, then incubated at hypoxic condition (5% O₂) for indicated time. Nuclear (N) and cytoplasm (C) were separated. HIF-1α, Lamin B (marker for nucleus) and GAPDH (marker for cytoplasm) were examined by Western blot using specific antibodies. (D) Quantification of nuclear HIF-1α protein levels in HepG2/DDP cells from 2 experiments shown in (C). Data were normalized to time 0. Student’s t-test: *P<0.01. (E) Nrf2 knockdown blocked the sustained induction of HIF-1α target gene GLUT-1 in HepG2/DDP cells by hypoxia. Cells were transfected with Nrf2-specific siRNA for 24 hours, then incubated at hypoxic condition (5% O₂) for indicated time. Total mRNA was extracted and reversed transcribed. GLUT-1 cDNA was examined by RT-qPCR. Data from 3 experiments were plotted and analyzed with Student’s t-test: ns, not significant, **P<0.001. (F) KEAP-1 knockdown blocked the rapid degradation of HIF-1α in HepG2 under hypxia. siRNA knockdown, nuclear purification and Western blot was performed as shown in (C). (G) Quantification of Western blot signals in (F). Data were normalized to time 0. Student’s t-test was performed to evaluate the statistical significance. *P<0.01. (H) KEAP1 knockdown blocked GLUT-1 down-regulation in HepG2 cells under hypoxia. Data from 2 repeats normalized to time 0. Student’s t-test: *P<0.01, **P<0.001.