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

FIGURE 1.

FIGURE 1

Translation from the HCV IRES in Huh-7 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 Huh-7 cells. Cells were transfected with in vitro synthesized HCV-Luc or EMCV-Luc mRNAs for 1 h and then incubated with either cycloheximide CHX (5 μg mL-1) or ARS, TG, SAL, or TM for a further 2 h. Percentage change is relative to that in the 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. (B) Inhibitor treatment induces eIF2 phosphorylation in Huh-7 cells. Cells were transfected with in vitro synthesized HCV-Luc RNA as above and then treated or not with ARS (100 μM), TG (5 μM), TM (40 μg mL-1), or SAL (200 μM) for 2 h. Proteins were resolved by SDS-PAGE and blots were probed with antibodies against 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. (C) Huh-7 cells were transfected with different in vitro transcribed reporter RNAs: HCV-Luc, EMCV-Luc, CrPV-Luc or Cap.βGlobin-Luc. After 1 h of transfection, cells were treated or not with ARS or TG, or with CHX, after which luciferase activity was measured. Bars represent the relative luciferase activity with non-treated control (inhibitor concentration = 0) set as 100%. The readings from CHX treatments were subtracted from all as a baseline. Error bars represent the standard error of the mean, n = 3. (A,C) 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. (D) Huh-7 cells were transfected with in vitro synthesized rep HCV-Luc mRNA. After 1 h of transfection, cells were treated with different concentrations of ARS or CHX (5 μg mL-1) for 2 h, after which luciferase activity was measured. Bars represent the relative luciferase activity with non-treated control (inhibitor concentration = 0) set as 100%. The readings from CHX treatments were subtracted 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 one-way ANOVA and a Tukey’s post hoc test, and is shown as: ∗p < 0.05, ∗∗∗p < 0.001.