Hemin induces NRF2 to bind to the γ-globin promoter and requires NRF2 for full induction of γ-globin. (A) Effect of tBHQ and hemin on NQO1 and γ-globin mRNA levels. K562 cells were treated with 25μM of tBHQ and 20μM of hemin for 12 and 24 hours before RNA isolation. (B) ChIP analysis of K562 cells treated with hemin for 0, 8, and 26 hours. Quantitative real-time PCR was performed on immunoprecipitated DNA with the use of primers that flank the ARE region in the γ-globin promoter. The NQO1 promoter was used as a positive control, and necdin promoter was used as a negative control. *P < .05 compared with untreated. Results presented as Enrichment over IgG, normalized to necdin. (C) Effect of NRF2 siRNA on hemin fold induction of NQO1 and γ-globin mRNA. siRNA specific for NRF2 was transiently transfected into K562 cells. At 24 hours later, cells were treated with tBHQ for 12 or 24 hours before RNA isolation. Control siRNA and a mock transfection (−) were used as controls. Results of real-time PCR are presented as fold induction. *P < .05 compared with control siRNA.