Induction of the Hri-eIF2αP-Atf4 signaling pathway and protection against oxidative stress in FL erythroblasts. (A) ROS levels. Hri^+/+ FL cells were treated with 25μM sodium arsenite. At times indicated, cellular ROS levels were measured by DCF fluorescence. (B) Hyperphosphorylation and activation of Hri. Hyperphosphorylation of Hri was examined by the slower migration in SDS-PAGE. eIF2α was used as a loading control. (C) Induction of the Hri-dependent Atf4 signaling pathway. Cells were treated with 5μM As. At times indicated, samples were analyzed for expression of eIF2αP, Atf4, Chop, and eIF2α proteins. (D) Relative expression of mRNAs of the Hri-Atf4 signaling pathway components. Atf4, Chop, Gadd34, and Crep mRNA levels from FL cells treated with 5μM As for 3 hours were analyzed by qPCR. Expression of each of the mRNA in untreated Hri^+/+ cells was defined as 1. Data are presented as mean ± SD (n = 3). P values denote the comparison between arsenite-treated Hri^+/+ and Hri^−/− samples; P < .001 for Atf4 and Gadd34; P < .005 for Chop. (E) ROS levels. Hri^+/+ and Hri^−/− FL cells treated with 5 or 25μM arsenite for 9 hours before ROS measurement.