Reduced ABCC4 activity enhances in vitro erythropoiesis of human CD34⁺ cells as well as erythropoiesis in WT and Ent1^−/− mice and enhances. (A) CD34⁺ cells from healthy donors were cultured in the presence or absence of MK-571 (100 μM), initiated 24 hours after CD34⁺ selection. GPA expression was monitored at day 3 of rEPO-induced erythroid differentiation and representative FACS histograms of control (black histogram) and MK571-treated progenitors (orange histogram) are presented, including percentages of GPA⁺ cells and GFMI (left). Shaded gray histograms correspond to unstained negative controls specifying negative gating. GMFI (middle) and the percentages of GPA⁺ cells (right) were quantified and means ± standard error of the mean (SEM) of 3 independent experiments are shown. *P < .05. (B) Erythroblast maturation was monitored at day 12 of differentiation in the presence or absence of MK-571 by α4-integrin/Band3 profiles of GPA^pos cells. Representative dot plots are shown (left), and the percentages of cells in each quadrant are indicated. Quantification of cells in the more immature α4-integrin^high/Band3^low and α4-integrin^med/Band3^med subsets as compared with the more mature α4-integrin^low/Band3^high subset is presented for 3 independent experiments (right). *P < .05. (C) Erythroblast enucleation was evaluated as a function of Syto16 nucleic acid staining and representative dot plots indicating the percentages of Syto16-enucleated cells (left) and quantification of enucleated cells in 3 independent experiments are presented (right). *P < .05. (D) Hematological parameters of WT and Ent1^−/− mice under control conditions or following a 6-day treatment with MK-571. *P < .05, **P < .01, ***P < .001, WT treated vs WT, Ent1^−/− treated vs Ent1^−/− untreated. (E) Quantification of CMP, MEP, and GMP in BM of WT and Ent1^−/− mice in the absence or presence of MK-571 treatment (10 mg/kg every other day for 1 week, n = 4-6). (F) Frequency of Lin⁻c-kit⁺ cells in BM and spleen of WT and Ent1^−/− mice treated or not with MK-571. (G) Quantification of the frequencies of Ter119⁺ and Gr-1⁺ cells in BM of untreated and MK-571-treated WT and Ent1^−/− mice are presented (n = 4-6). *P < .05, **P < .01.