Mitochondrial metabolism is required for murine erythroblast enucleation. (A) Mitochondrial abundance and MMP was measured with MitoTracker Green (MTG) and TMRE, respectively, in erythroblasts. Gating strategy to identify erythroblasts (G1-G3), reticulocytes (G4), and RBCs (G5; top). Histograms display MTG (middle) and TMRE (bottom) intensities. (B) Quantification of the average intensities of MTG (top), TMRE (middle), and normalized TMRE to MTG (bottom) in distinct erythroblast populations. Fluorescence values normalized to values of gate 1. TMRE levels were normalized to MTG levels to determine the mitochondrial activity of the total mitochondria present within each erythroblast population. TMRE/MTG was calculated for each gate and values normalized to gate 1 value. (C) Oxygen consumption rate (OCR) as measured by the seahorse XF-96 of nucleated erythroblasts, reticulocytes, and RBCs undergoing FACS. Oligomycin (oligo), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone and antimycin A (Rot/AA) were added at the indicated time points. (D) Gating strategy for ex vivo enucleation assay. Orthochromatic erythroblasts (G3) undergoing FACS and cultured in StemSpan + 2% FBS were allowed to enucleate. Enucleation rates were assessed based on DRAQ5 staining vs forward scatter (FSC) properties. (E) ATP levels measured from cell lysates treated with increasing concentrations of the electron transport chain (ETC) uncoupler BAM15 at the 4-hour time point. (F) Flow plots of enucleation in response to various inhibitors at indicated concentrations on ETC after 18 hours in the ex vivo enucleation assay (left) and quantification of enucleation rates normalized to dimethyl sulfoxide (DMSO; right panel). CCCP pulse for 2 hours was followed with by washout. Mean ± standard error of the mean (n ≥ 3). *P < .05, **P < .01, ***P < .001 by 1-way analysis of variance followed by Tukey multiple comparisons test (B,E-F). BM, bone marrow; n.s., not significant.