Irg1^–/– mice exhibit a defect in recovery from PHZ-induced acute hemolytic anemia. WT mice were injected with PHZ (100 mg/kg body weight of mouse) and analyzed on the indicated days. (A) Nrf2 protein expression in the spleen on the indicated days was determined by western blot analysis. β-Actin is shown as a loading control. Corresponding western blots are shown in supplemental Figure 5B. (B) Metabolomic analysis of itaconate levels in Kit⁺ SEPs isolated from the spleen on days 0, 1, and 3 after PHZ treatment (n = 5 per day). (C) qRT-PCR analysis of mRNA expression of Nrf2 target genes, Nqo1 and Gclm, in the spleen of WT and Irg1^–/– mice on the indicated days of recovery from PHZ-induced anemia (n = 3 per time point; unpaired t test). (D) qRT-PCR analysis of select erythroid genes in the spleen of WT and Irg1^–/– mice on the indicated days of recovery from PHZ-induced anemia (n = 3 per time point; unpaired t test). (E) Number of stress BFU-E in the spleen (left) and hematocrit (right) on the indicated days in WT and Irg1^–/– mice treated with PHZ (n = 3 per time point; unpaired t test). Gclm, glutamate-cysteine ligase, modifier subunit.