D-2HG directly inhibits the enzyme activity of OGDH. (A) Heat map displaying relative quantitation of Krebs cycle intermediates in Ter-119⁺ erythroblasts from BM and spleen of control and IDH1-KI mice as determined by LC-MS/MS. As indicated, the colors represent the relative abundance (fold change) of these metabolites in IDH1-KI vs control mice (n = 6). (B) Relative mRNA levels of the indicated Krebs cycle enzymes 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. (C) Immunoblot to detect OGDH, Suclg1, and succinate dehydrogenase complex subunit B (SDHB) 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. (D) Drug affinity responsive target stability assays (see Materials and methods) to detect the interactions of OGDH protein with α-KG (top) or D-2HG (bottom) and Suclg1 protein with D-2HG (bottom) in nontransfected K562 cells. FH and CS proteins were used as negative control. Data are representative of 3 trials. (E) Time course of enzymatic activity of OGDH (as determined by using a colorimetric assay kit) in mouse BM Ter-119⁺ erythroblasts (top) or nontransfected K562 cells (bottom) that were treated with the indicated concentrations of D-2HG. Data are representative of 3 trials. (F) Top, microscopic views of representative May-Grünwald-Giemsa–stained K562 cells treated with or without cell permeable octyl-D-2HG. Scale bar, 20 μm. Bottom, corresponding cell pellets for the cultures in the top panels. (G) Flow cytometric determination of the erythroid marker CD235a expression in K562 cells treated with or without octyl-D-2HG. Data are representative of triplicate assays. (H) Quantitation of heme content in the cells in panel G. (I) Immunoblot to detect hemoglobin proteins in the cells in panel G. Data are the mean ± SEM. *P < .05; **P < .01; ***P < .001.