Loss of NRF2 function produced severe splenomegaly and inflammation in SCD mice. (A) The weight (left) and size (right) of spleens from NRF2 wild-type (^+/+), heterozygote (^+/−), and knockout (^−/−) SCD mice were determined. (B) Histologic section of spleen tissue stained with hematoxylin and eosin is shown; scale bars: (i and ii), 100 µm; (iii and iv), 40 µm. (C) Using 2'-7'-dichlorodihydrofluorescein diacetate staining and flow cytometry, ROS levels in RBCs were determined (n ≥ 6); RBC gating was performed as described in supplemental Methods (supplemental Figure 2). (D) Western blot was completed to determine the expression of antioxidant proteins in spleen whole-tissue extracts for adult SCD/NRF2^+/+ mice (red bars) and SCD/NRF2^−/− mice (blue bars) (n = 3 per group) (top), and quantitative data were generated (bottom); β-actin was used as a loading control. (E) Western blot was completed as in panel D for the following: intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), and vascular endothelial growth factor (VEGF-A) (top), and quantitative data analyzed (bottom). (F) The mRNA levels of the various genes shown were measured by qRT-PCR in spleen cells isolated from 2- to 3-month-old mice (n = 6 per group). Red bars, C57BL/6 mice; blue bars, SCD/NRF2^+/+ mice; light green bars, SCD/NRF2^−/− mice. The level of mouse 18s ribosomal RNA was used as an internal control. **P < .01; *P < .05. CAT, catalase; GCLC, γ-glutamylcysteine ligase catalytic subunit; HMOX1, heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1; qRT-PCR, quantitative reverse transcription polymerase chain reaction.