Impaired in vitro and in vivo functionality of Pyronin^low/Hoechst^low GMPs from GATA-2^+/− marrow. GMPs from GATA-2^+/+ and GATA-2^+/− bone marrow were isolated by cell sorting and were subsequently stained with the RNA dye Pyronin Y (PY) and DNA dye Hoechst 33342 (Hst). Flow cytometry was performed to distinguish the PY^lo or PY^high in the G₀/G₁ fraction (Hst^low). Cells residing in PY^low/Hst^low or PY^high/Hst^low were sorted and plated into colony-forming medium containing myeloid, megakaryocyte, and erythroid growth factors. Three replicates were used per genotype in each experiment. Colony formation was scored on day 10 (A) (B) GMPs from GATA-2^+/+ and GATA-2^+/− bone marrow were isolated by sorting and were subsequently stained with the DNA dye Hoechst 33342 (Hst) to enable discrimination of G₀/G₁ and S/G₂/M populations The S/G₂/M population was sorted and plated into colony-forming medium containing myeloid, megakaryocyte, and erythroid growth factors. Three replicates were used per genotype in each experiment. Colony formation was scored on day 10 (C). Data from multiple experiments are displayed (A-C) (PY^low/Hst^low, n = 5; G, P = .92; M, P = .06; GM, P = .002; PY^high/Hst^low, n = 5; G, P = .47; M, P = .85; GM, P = .83; S/G₂/M, n = 6; G, P = .71; M, P = .97; GM, P = .99). In vivo functionality was assessed by competitive transplantation of PY^low/Hst^low or PY^high/Hst^low GMPs from each genotype (B6SJL-CD45.1) with C57BL/6-CD45.2 Lin-Sca-1⁺CD117⁺CD34⁻ competitor stem cells into C57BL/6-CD45.2–irradiated recipients. Eight days after transplantation, the peripheral blood of the recipient mice were analyzed for the contribution of donor CD45.1 within the Gr-1⁺Mac-1⁺ compartment by flow cytometry. Data from multiple experiments are displayed for PY^low/Hst^low (D) (n = 3; P = .042) and PY^high/Hst^low (E) (n = 3; P = .31). ** indicates statistical significance. Error bars indicate SEM. Statistical analysis was performed using the paired Student t test.