tBHQ increases γ-globin mRNA and NRF2 binding to the γ-globin promoter in primary human erythroid cells. Human CD34⁺ peripheral blood cells were treated with tBHQ during in vitro erythroid differentiation. The effects of increasing concentrations of tBHQ were determined for (A) cell expansion and (B) cytospins from untreated and cells treated with 5μM tBHQ at end of differentiation (day 20). (C) NQO1 gene expression at specific time points during differentiation relative to untreated n = 3, (D) γ/γ+β mRNA at specific time points during differentiation relative to untreated, n = 3. (E) tBHQ dose response on γ, β and γ/(γ + β) mRNA expressed as the AUC for each dose n = 3, 5-Aza was used as a positive control for γ-globin mRNA induction (panels C and D). Error bars represent ± 1 SD. (F) ChIP analysis of CD34⁺ cells on day 13 of differentiation treated with 0, 1, and 5μM tBHQ. 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. n = 2, *P < .05 compared with untreated. Photomicrographs were taken using a Nikon eclipse 80i camera at 10× magnification and Qcapture v2.7.3 software.