HRI-ISR is activated in ID and is necessary to promote differentiation and mitigate ROS. (A) HRI-eIF2αP signaling and (B) ATF4 expression in erythroid precursors from Wt, Hri^−/−, eAA, and Atf4^−/− mice. (C) Differentiation stages of Ter119⁺ cells from BM and spleen (Spl). +exp and –exp denote with or without splenic erythroid expansion, respectively, of Atf4^−/−–Fe mice (supplemental Figure 2C-E). (D-E) Representative ROS histograms for Ter119⁺ populations from eAA and Atf4^−/− mice. Wt–Fe (green shade), eAA+Fe (blue line), eAA–Fe (red line), and Atf4^−/−–Fe (red line) in BM, spleen, and blood samples. Numbers of mice used in (C): Wt+Fe, n = 34; Hri^−/−+Fe, n = 8; eAA+Fe, n = 6; Atf4^−/−+Fe, n = 4; Wt–Fe, n = 31; Hri^−/−–Fe, n = 9; eAA–Fe, n = 11; Atf4^−/−–Fe +exp, n = 5; Atf4^−/−–Fe, –exp, n = 5. Ret, reticulocyte.