Mitochondrial activity identifies subpopulations of murine orthochromatic erythroblasts at distinct stages of maturation. (A) Flow plot displaying the gating used to segregate 20% of the lowest and highest MMP orthochromatic erythroblasts (TMRE⁻ cells were excluded). (B) Flow plot displaying MMP-low and -high orthochromatic fractions plotted onto the total TER119⁺ parent population (also supplemental Figure 6Bi). (C) Histogram comparing CD44 expression between MMP-low and -high fractions (left) and quantification based on average intensities (right). (D) Ex vivo enucleation assay kinetics over 3 time points (3, 5, and 18 hours). Cell counts of MMP-low and -high fractions normalized to cell number at the start of plating. (E) Cell cycle analysis of MMP-low and -high fractions undergoing FACS with Ki67 and DAPI staining; gating strategy (left) and quantification (right). (F) Confocal imaging of DAPI-stained MMP-low and -high orthochromatic erythroblast fractions (top) and quantification of nucleus perimeter (middle) and area (bottom) as determined by ImageJ (bar, 5 μm). (G) Western blot analyses of acetylation marks H3K27 and H3K9 comparing MMP-low and -high fractions undergoing FACS; 1 representative blot of 3 independent experiments is shown (top); quantification (bottom). (H) Confocal images of H3K9me3 (top) and quantification of H3K9me3 intensity (bottom). Note arrows show H3K9me3 in DAPI bright regions of MMP-high and mislocalized H3K9me3 in MMP-low cells; mean ± standard error of the mean (n ≥ 3 each from 3 mice except for H3K9me3 from 2 experiments with 3 mice each; bar, 5 μm). *P < .05, **P < .01, ***P < .001 by Student t test (C-H).