Nrf2 and L2HG regulate ferroptosis in SCD. (A) RNA-seq determined that ROS and ferroptosis significantly affect signaling pathways in Nrf2 ablated SCD mouse bone marrow Ter119⁺ cells (n = 3). (B) Immunoblotting showing the expression of Nrf2 targeted proteins that were involved in antioxidant (Nqo1, Cat, Gclc, and Gstt1) and ferroptosis stress responses (Hmox1, Fech, Tfrc, Ftl1, Fth1, and Slc7a11) in SS/Nrf2^+/+ and SS/Nrf2^–/– spleens. The transcription factor Tbp and β-actin are loading controls. (C) Distinct levels of heme, iron, and bilirubin in the serum, spleen, and liver of SS/Nrf2^+/+ and SS/Nrf2^–/– mice. (D) Iron levels in SS/Nrf2^+/+ and SS/Nrf2^–/– mouse spleens and liver tissues by Prussian Blue staining (relative staining intensity) (right). (E-F) NADPH:NADP⁺ ratio (E), GSH and GSSG levels, and GSH:GSSG ratio (F) in the spleens of SS/Nrf2^+/+ and SS/Nrf2^–/– mice. (G) Flow cytometry analysis of lipid peroxidation levels in spleen Ter119⁺ cells of SS/Nrf2^+/+ and SS/Nrf2^–/– mice after stained with C11-Bodipy. (H) Immunoblotting showing the lipid peroxidation levels of 4HNE production in the spleen of SS/Nrf2^+/+ and SS/Nrf2^–/– mice. Data represent mean ± SD of 3 biological replicates. ∗P < .05; ∗∗P < .01. In panels B-C, a one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test was used for statistical analysis. 4HNE, 4-hydroxynonenal; NADPH, reduced NADP; Tbp, TATA-box binding protein.