Reduced heme catabolism contributes to increased ROS and death in IDH1-mutant erythroid cells. (A) Left, representative flow cytometric analysis of ROS levels (as determined by CMH₂DCFDA staining) in Ter-119⁺ erythroblasts from BM and spleen of control and IDH1-KI mice. Right, quantitation of the percentages of ROS positive BM and spleen Ter-119⁺ erythroblasts in the left panels. Data are the mean ± SEM (n = 3). (B) HPLC/LC-MS determination of relative levels of reduced NADP (NADPH), NADP⁺, NADH, and NAD⁺ in erythroblasts from BM and spleen of control and IDH1-KI mice. Data are the mean ± SEM (n = 6). (C) HPLC/LC-MS determination of relative levels of bilirubin and biliverdin in Ter-119⁺ erythroblasts from BM and spleen of control and IDH1-KI mice. Data are the mean ± SEM (n = 6). (D) Relative HO-1 mRNA levels in Ter-119⁺ erythroblasts from BM and spleen of control and IDH1-KI mice. Data are the mean ± SEM (n = 6). β-actin, endogenous control. (E) Immunoblot to detect HO-1 protein in Ter-119⁺ and Ter-119^– nucleated cells from BM and spleen of control and IDH1-KI mice. Data are representative of 3 trials. (F) Immunoblot to detect the indicated HO-1 protein in cultures of K562 cells that expressed WT or mutant IDH1 (n = 3). β-actin, loading control. (G) Left, propidium iodide (PI) and FITC-Annexin V staining to detect cell death in K562 cells in panel F treated with or without bilirubin, biliverdin, or acetylcysteine, as indicated. Data are representative of 3 trials. Right, quantitation of the percentages of apoptotic and necrotic cells in the cultures in the top panel (n = 3). (H) CMH₂DCFDA determination of ROS in the cells in panel G. Data are representative of 3 trials. *P < .05; **P < .01; ***P < .001.