siRNA knockdown of NRF2 or mutation of the ARE element inhibit tBHQ induction of γ-globin gene expression. siRNA specific for NRF2 was transiently transfected into K562 cells. At 24 hours later, cells were treated with tBHQ for 12 hours before RNA isolation. Control siRNA and mock transfection (−) were used as controls. (A) Western blot analysis of total cellular NRF2 protein after siRNA transfection and tBHQ treatment. (B) Effect of NRF2 siRNA on tBHQ induction of NQO1 and γ-globin mRNA. Results of real-time PCR are presented as relative mRNA expression normalized to untreated control siRNA. (C) ARE element is required for tBHQ induction. Forty micrograms of WT or mutant ARE constructs were electroporated with 10 μg of pCMVGLuc control vector into K562 cells. tBHQ was added to a final concentration of 25μM at 24 hours after transfection and harvested at 48 hours. WT-untreated vector was set to 100%. Transfection efficiency was controlled for by normalizing to pCMVGLuc signal. Error bars represent ± SEM. P values are calculated relative to untreated controls. *P < .05.