Effects of human IDH1-R132H overexpression. (A) IDH1 protein expression in uninjected and IDH1-WT– or IDH1-R132H–injected embryos at 24 hpf. The numbers indicate the relative band intensity as quantified by Image J software and normalized to levels of β-actin. (B) Representative gas chromatographs from IDH1-WT– or IDH1-R132H–injected embryos with or without AGI-5198 treatment. The spikes represent the MTBSTFA-derivatized intracellular metabolites eluting between 31.0 and 34.0 minutes, including aspartate (Asp), 2-HG, and glutamate (Glu). Black arrows indicate the expected elution time of 32.6 minutes for MTBSTFA-derivatized 2-HG. Metabolite abundance refers to GC-MS signal intensity. Quantitation of 2-HG signal intensities relative to the intrasample glutamate signals of 3 different experiments was shown. (C) 5-hydroxymethylcytotsine (5-hmC) was reduced upon IDH-R132H expression. Expression of early myeloid progenitor marker pu.1 (D), and late myelomonocytic differentiation markers l-plastin (E), mpo (F), and mpeg1 (G) at 24 hpf in embryos injected with complementary DNA encoding IDH1-WT, or IDH1-R132H, with or without AGI-5198 (10 μM) treatment starting from bud stage. The red arrows indicate myeloid cells on the yolk sac. (H-K) The cell numbers of pu.1 (H), l-plastin– (I), mpo- (J), and mpeg1- (K) positive cells per embryos. (L) Fluorescent microscopy of Tg(mpo:gfp) embryos at 24 hpf showing increased gfp⁺ cells in embryos injected with IDH1-R132H compared with IDH1-WT. The inset (L′) showed the morphology of gfp⁺ cells in dissociated Tg(mpo:gfp) embryos at 36 hpf. (M,N) Percentage of mpeg1:GFP⁺ and mpo:gfp⁺ cells in uninjected control–, IDH1-WT–, and IDH1-R132H–injected embryos at 24 hpf as enumerated by flow cytometry (representative results are shown in supplemental Figure 11) in 3 different experiments. n.s., not significant. Bars represent 250 μm and ×600 magnification in panel (L′).