Characterization of erythroid phenotypes of eAA and Atf4^−/−mice in ID. (A) Generation of eAA mice by crossing AATg mice with EpoRCre⁺ mice. (B) Defective eIF2αP in Ter119⁺ cells of eAA mice. Sorted Ter119⁺ erythroblasts (populations I+II) and Ter119^– (supplemental Figure 1A-B) from BM were used. (C) Complete blood cell count (CBC) of Wt, Hri^−/−, eAA, and Atf4^−/− mice under both +Fe and –Fe conditions. (D) Wright-Giemsa stained blood smears. Arrows indicate globin inclusions. Photographs were taken by using a Leitz optical microscope with PixeLINK Capture software. (E) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assay of insoluble globin precipitates from blood samples. Precipitated globin protein in 2000g pellets of equal numbers of blood cells (1 × 10⁷) were analyzed. (F) Spleen weights as percentage of body weights. (G) Serum Epo levels. (H) Correlation of serum Epo to hemoglobin levels. P values denote the comparison between Wt and mutant mice under –Fe conditions. *P < .05, **P < .01, ***P < .001. Numbers of mice used in (C and F): Wt+Fe, n = 25; Hri^−/−+Fe, n = 5; eAA+Fe, n = 6; Atf4^−/−+Fe, n = 11; Wt–Fe, n = 22; Hri^−/−–Fe, n = 6; eAA–Fe, n = 11; Atf4^−/−–Fe, n = 10. Numbers of mice used in (G): Wt+Fe, n = 11; Hri^−/−+Fe, n = 8; eAA+Fe, n = 5; Atf4^−/−+Fe, n = 4; Wt–Fe, n = 9; Hri^−/−–Fe, n = 9; eAA–Fe, n = 10; Atf4^−/−–Fe, n = 6. Numbers of mice used in (H): Wt–Fe, n = 6; Hri^−/−–Fe, n = 6; eAA–Fe, n = 10; Atf4^−/−–Fe, n = 6. BW, body weight; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.