NRF2 loss decreased γ-globin gene transcription in SCD mice during gestation. SCD mice with homozygous NRF2 knockout (SCD/NRF2^−/−) were established by crossbreeding Townes SCD mice¹³  and NRF2 knockout (NRF2^−/−) mice¹⁴  (supplemental Methods; supplemental Table 1). (A) Hematological indices of 2- to 3-month-old SCD/NRF2^+/+ (red bars) and SCD/NRF2^−/− mice (blue bars) were analyzed by automated complete blood counts and differentials. (B) Expression of human γ-globin and adult β^S-globin genes was monitored by qRT-PCR in embryonic day 13.5 and embryonic day 18.5 fetal livers and adult spleen and bone marrow (n = 6-8 mice per group); the γ/(γ+β^S) mRNA ratios were calculated. Red circles, SCD/NRF2^+/+ mice; blue circles, SCD/NRF2^−/− mice. (C) Bone marrow hematopoietic progenitors were isolated from transgenic mice and used for analysis of the different proteins by flow cytometry (supplemental Methods). Representative scatter plots are shown. (D) The levels of HbF and HbS were determined by western blot analysis of whole-tissue protein extracts isolated from adult SCD/NRF2^+/+ mice (red bars) and SCD/NRF2^−/− mice (blue bars) spleens (n = 3) (top), and quantitative data were generated (bottom); β-actin was used as a loading control. (E) Flow cytometry analysis determined the percentage of HbF-positive RBCs (F-cells) in the peripheral blood stained with fluorescein isothiocyanate–conjugated HbF antibody; RBC gating was performed as described in supplemental Methods (supplemental Figure 2). Representative dot plots for F-cell distribution are shown (left), and the percentage of F-cells was quantified (right) (n = 8). Red circles, SCD/NRF2^+/+mice; blue circles, SCD/NRF2^−/− mice. C57BL/6 mice were non–sickle cell controls. (F) The level of RBC sickling in the peripheral blood was quantified under normoxic (21% O₂) and hypoxic (1% O₂) oxygen conditions (left); data represent the mean ± standard deviation (n = 6-7). Representative images under bright field are shown at ×600 magnification (right). *P < .05. qRT-PCR, quantitative reverse transcription polymerase chain reaction.