Mx1-Cre;Atf4^fl/fl mice experience severe macrocytosis. (A) Representative images of femurs and PB smears. (B) Routine blood parameters of fl/fl and Δ/Δ mice (n = 4-5 mice). (C) Survival curves of fl/fl and Δ/Δ mice treated with 25 μg/g pIpC delivered on alternate days for a total of 3 doses. (D) Percentages of terminal erythroid cell populations in BM (n = 3-4 mice). (E) Representative flow cytometry dot plots of CD44 expression vs forward scatter (FSC), after gating on TER119⁺ cells (left) and quantification of terminally differentiated erythroid subsets in fl/fl and Δ/Δ mice (right) (n = 4-5 mice). (F) Quantitative results from BFU-E colony formation assays performed using 3 × 10³ cKit⁺ BM cells from fl/fl and Δ/Δ mice cultured in MethoCult SF M3436 methylcellulose-based medium for 10 to 14 days (n = 4 mice). (G) Quantitative results (left) from CFU-E colony formation assays performed using 3 × 10³ cKit⁺ BM cells from fl/fl and Δ/Δ mice cultured in MethoCult SF M3334 methylcellulose-based medium for 48 hours and representative images from 3 independent experiments (right). Scale bar, 25 μm (n = 4 mice). (H) Representative flow cytometry dot plots (bromodeoxyuridine [BrdU]/Hoechst) of MEP cells (left) and a graph showing the percentage of BrdU⁺ MEP cells (right) (n = 3-4 mice). (I) Percentages of MEP cells in different cell cycle phases (n = 3-4 mice). (J) Quantitative results (left) from CFU-E colony formation assays performed using 500 fresh BM MEP sorted from fl/fl and Δ/Δ mice and cultured in MethoCult SF M3334 methylcellulose-based medium for 48 hours (right) (n = 3 mice). (K) Quantitative results (left) from BFU-E colony formation assays performed using 1000 fresh BM MEP cells sorted from fl/fl and Δ/Δ mice and cultured in MethoCult SF M3436 methylcellulose-based medium for 2 weeks (right), (n = 3 mice). Data represent the mean ± SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; unpaired 2-tailed Student t test. HGB, hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume.