Figure 3

Effects of the phospho-eIF2α/ATF4 pathway on NMD activity under ER stress. NS, non-silencing. (a,c) Effects of Atf4 (a) or Perk (c) knockdown on Hnrnpl_NMD mRNA expression were analyzed by RT-qPCR. Total RNA was extracted from untreated cells or following treatment with 0.2 μg/ml TPG for 16 h. The histogram depicts Hnrnpl_NMD mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated NS siRNA-transfectants. Values represent mean ± SE of three separate experiments. *^,***Significantly different from TPG-untreated cells by one-way ANOVA followed by Bonferroni’s multiple comparison test (*p < 0.05, ***p < 0.001). ^###Significantly different from TPG-treated NS siRNA-transfectants by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.001). (b,d) Effects of Atf4 (b) or Perk (d) knockdown on NMD components expression. Cell lysates were prepared 16 h after exposure to 0.2 μg/ml TPG. Cropped blots are shown; all gels were run under the same experimental conditions. Raw data are shown in Supplementary Fig. 3b and d with dividing lines and with quantitative data. (e) Effects of eIF2α phosphorylation on Hnrnpl_NMD mRNA expression were analyzed by RT-qPCR. WT, cell line transfected with wild-type eIF2α plasmid. SA, cell line transfected with mutant eIF2α-SA plasmid. Endogenous eIF2α was knocked down by transfection with synthetic siRNA against eIF2α (si-Ei). Total RNA was extracted from untreated cells or following treatment with 0.2 μg/ml TPG for 16 h. The histogram depicts Hnrnpl_NMD mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated WT control. Values represent mean ± SE of three separate experiments. ***Significantly different from TPG-untreated cells by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.001). ^###Significantly different from TPG-treated WT cell line by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.001). (f) Effect of eIF2α phosphorylation on NMD components expression under mild ER stress. Cells are as shown in (e). Cell lysates were prepared 16 h after exposure to 0.2 μg/ml TPG. Cropped blots are shown; all gels were run under the same experimental conditions. Raw data are shown in Supplementary Fig. 3f with dividing lines and with quantitative data.