FIG 2.

Phosphorylation of eIF2α during DENV4 or ZIKV infections is PKR dependent and IFN independent. (A) Characterization of A549 PKR^−/− by immunoblot of unstimulated and stimulated cells with 100 IU/mL of IFN-α2a for 12 hours demonstrating successful PKR deletion. (B) Characterization of A549 IFNAR^−/−/PKR^−/− after stimulation with 100 IU/mL of IFN-α2a for 12 hours demonstrating the deletion of PKR on the parental IFNAR^−/− cells. (C) Flow cytometry analysis for quantification of cells expressing p-eIF2α within the total population of the indicated lineages of A549 cells infected with DENV4 (MOI 2) at 24 h.p.i. and (D) same experiment using ZIKV (MOI 3). (E) Flow cytometry analysis for quantification of cells expressing p-eIF2α within the total population A549 WT and PKR^−/− cells infected with DENV4 (MOI 2) or ZIKV (MOI 3) or treated with thapsigargin 2 µM for 45 minutes in the presence or absence of the PERK inhibitor GSK2656157 (5 µM) for 24 hours and (F) immunoblot analysis of cell extracts from the same experiment resolved in denaturing SDS-PAGE. Representative image of two independent experiments. In column charts, bars represent the mean ± standard error of the mean from three independent experiments. Statistical analysis was performed by one-way analysis of variance, followed by Tukey’s test for multiple comparisons. ** ≤ 0.05, *** ≤ 0.01, **** ≤ 0.0001. DKO, double IFNAR//PKR/− knockout; IFN, interferon; ns/no markup, no statistical difference.