Nrf2 activation reverses SCD ferroptosis stress. (A) Levels of heme, nonheme iron, and bilirubin (total and indirect) in SCD mice before and after chronic DMF or vehicle 0.08% hydroxyethyl cellulose (Veh) treatment. (B) Immunoblotting showing the expression of antioxidants (Nqo1 and Gclc), ferroptosis stress response proteins (Hmox1, Fth1, Ftl1, and Slc7a11), and L2hgdh in the spleen Ter119⁺ cells of SCD mice. (C) L2HG levels in spleen Ter119⁺ cells from SCD mice. (D) Immunoblotting showing histone methylation in the histone extracts of spleen Ter119⁺ cells of SCD mice. (E) ChIP-PCR analysis of the association of Nrf2, Tbp, RNA Pol II, and H3K27Me3 with the promoter regions of antioxidant (Nqo1, Cat, Gclc, and Gstt1), and ferroptosis stress response genes (Hmox1, Fth1, Ftl1, Slc7a11, and Slc40a1) in the SCD mouse spleen Ter119⁺ cells. Data represent mean ± SD (n = 3-10). ∗P < .05. (F) Schematic depicting the Nrf2 function in SCD EPs. ROS stress and heme accumulation from chronic hemolysis activate Nrf2 and subsequently L2hgdh expression to reduce the levels of L2HG. Reduced L2HG increases the enzymatic activities of α-ketoglutarate–dependent hydroxylases such as KDM and thus decreases global histone methylation, which further leads to the upregulated expression of antioxidant and ferroptosis-related genes to protect against heme and ROS stress. For panels B and E, a one-way ANOVA with Bonferroni multiple comparison test was used for the statistical analyses. Veh., vehicle.