K3 is crucial for EBI formation and EB maturation in the BM. (A) Total BM cells per femur. ∗P < .05; K3 mutant vs K3WT strains; n = 5 mice per strain. (B) Flow cytometric analysis of BM EB populations. Representative histograms of Ter119⁺ erythroid cells (top), which were further divided to EB progenitor populations I to VI, based on their size (forward side scatter) and CD44 expression (bottom). (C) Statistical comparison of erythroid Ter119⁺ cell quantity in mouse BM expressed as a percentage of BM cells, as shown in panel A (top). ∗P < .05; K3 mutant vs K3WT strains; n = 5 mice per group. (D) Statistical comparison of cell quantities at each stage of erythroid differentiation expressed as a percentage of Ter119⁺ cells. ∗P <.05; ∗∗P < .01; K3 mutant vs K3WT strains, n = 5 mice per group. (E) Representative images of reconstituted EBIs from single-cell suspensions of mouse BM labeled for macrophage Alexa Fluor-488 F4/80 (green) and erythroid Alexa Fluor 568-Ter-119 (red). The images were acquired using a Leica DM2500 confocal microscope with an ACS APO 63×/1.30 oil objective lens and the LAS-X software. The images were processed with Adobe Photoshop CC; scale bar, 23 μm. (F) Numbers of EBIs per field formed from mouse BM single-cell suspensions as described in “Materials and Methods.” ∗P < .01; K3 mutant vs K3WT strains; n = 10 mice per strain. (G) Representative images of BM smears stained for erythroid Ter119 (red-Alexa Fluor 568) and macrophage F4/80 (green-Alexa Fluor 488) markers. The images were taken with a Leica DM2500 confocal microscope equipped with an ACS APO 10×/0.3 objective lens and the LAS-X software, and processed using the Adobe Photoshop CC software; scale bar, 146 μm. (H) Representative histograms of F4/80⁺ macrophages quantified in mouse BM via flow cytometry. (I) Statistical comparison of data shown in panel G. ∗P < .01; K3 mutant vs K3WT strains; n = 5 mice per strain.