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. 2018 Feb 13;9:207. doi: 10.3389/fmicb.2018.00207

FIGURE 5.

FIGURE 5

Translation from the HCV IRES in HAP1 WT cells is resistant to the action of eIF2 inhibitors. (A) Translation from the IRES of HCV or EMCV as measured by luciferase activity in response to eIF2 inhibitor treatment in HAP1 WT cells. Cells were transfected with HCV-Luc or EMCV-Luc mRNAs for 1 h and then incubated with either CHX (5 μg mL-1) or ARS, TG, TM, or SAL for a further 2 h. Percentage change is relative to non-treated control (inhibitor concentration = 0). The readings from CHX treatments were subtracted from all as a baseline. Error bars represent the standard error of the mean, n = 3. Statistical significance of the differences between treated samples compared to control was calculated with two-way ANOVA and a Bonferroni post hoc test, and is shown as: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (B) Inhibitor treatment induces eIF2α phosphorylation in HAP1 WT cells. Cells were transfected for 1 h and then treated or not with either ARS (200 μM), TG (5 μM), TM (40 μg mL-1) or SAL (50 μM) for 2 h. Proteins were resolved using SDS-PAGE and then samples were probed with antibodies for phospho-eIF2α and total eIF2α. Shown is a representative blot from three independent experiments. The phosphorylation of eIF2α induction rate was evaluated by normalizing the raw value of P-eIF2α to that of total eIF2α as shown in the bar graph.