Figure 4

Effects of eIF2α phosphorylation on mTORC1 signaling. 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). (a) RT-qPCR analysis of Mtor mRNA. Total RNA was extracted from untreated cells or following treatment with 0.2 μg/ml TPG for 16 h. The histogram depicts Mtor 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 WT cell line by one-way ANOVA followed by Bonferroni’s multiple comparison test (^##p < 0.01, ^###p < 0.001). (b) Western blotting analysis for phospho-4EBP1 (p-4EBP1). Total 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 were shown in Supplementary Fig. 4b with dividing lines and with quantitative data. (c) Effect of the mTOR activator MHY1485 (MHY) on expression of p-4EBP1. SA cells were treated with the indicated concentration of MHY for 5 h before cell preparation. Total 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. 4c with dividing lines and with quantitative data. (d) Effect of mTOR activator MHY1485 (MHY) on NMD activity. Expression of Snhg1 mRNA was evaluated by RT-qPCR. Total RNA was extracted from SA cells 16 h after exposure to 0.2 μg/ml TPG. SA cells were treated with the indicated concentration of MHY for 3 h before RNA extraction. The histogram depicts Snhg1 mRNA normalized to Actb mRNA presented as the fold-increase over MHY- and TPG-untreated control. Values shown are the mean ± SE of three separate experiments. ^###Significantly different from TPG-treated MHY-non-treated cells by one-way Welch’s t-test (p < 0.001).