Journal: Scientific Reports

Article Title: Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

doi: 10.1038/s41598-018-38015-2

Figure Lengend Snippet: Effects of depleting NMD effectors on NMD-sensitive and NMD-non-sensitive isoform mRNA expression under mild ER stress. NS, non-silencing. ( a , c ) RT-qPCR analysis of NMD-sensitive mRNA of Hnrnpl_NMD ( a ) and Tra2b_NMD ( c ). Total RNA was extracted from untreated cells or following treatment with 0.2 μg/ml TPG for 16 h. The histogram depicts Hnrnpl_NMD or Tra2b_NMD mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated NS siRNA-transfectants. Values shown are the 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 NS siRNA-transfectants by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.001). ( b , d ) RT-qPCR analysis of mRNA of NMD-non-sensitive Hnrnpl ( b ) or Tra2b ( d ) isoform using the same data set of NMD-sensitive isoform mRNA of panel a or c, to determine expression under mild ER stress. The histogram depicts each mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated NS siRNA-transfectants. Values shown are the 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).

Article Snippet: Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′) siRNA was designed using the i-Score Designer and asymmetric siRNA was synthesized (GeneDesign, Inc.).

Techniques: Expressing, Quantitative RT-PCR, Comparison

Journal: Scientific Reports

Article Title: Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

doi: 10.1038/s41598-018-38015-2

Figure Lengend Snippet: 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. 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. f with dividing lines and with quantitative data.

Article Snippet: Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′) siRNA was designed using the i-Score Designer and asymmetric siRNA was synthesized (GeneDesign, Inc.).

Techniques: Activity Assay, Knockdown, Expressing, Quantitative RT-PCR, Comparison, Phospho-proteomics, Transfection, Plasmid Preparation, Mutagenesis, Control

Journal: Scientific Reports

Article Title: Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

doi: 10.1038/s41598-018-38015-2

Figure Lengend Snippet: 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. 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. 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).

Article Snippet: Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′) siRNA was designed using the i-Score Designer and asymmetric siRNA was synthesized (GeneDesign, Inc.).

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Control, Comparison, Western Blot, Expressing, Concentration Assay, Activity Assay, RNA Extraction

Journal: Scientific Reports

Article Title: Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

doi: 10.1038/s41598-018-38015-2

Figure Lengend Snippet: Effect of Perk knockdown ( a , b ), Atf4 ( c , d ) or the NMD components Smg-1 or Smg-7 ( e , f ) on mTORC1 signaling. NS, non-silencing. ( a , c , e ) Analysis of Mtor mRNA 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 Mtor mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated NS siRNA-transfectants. Values shown are the 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 TPG-treated NS siRNA-transfectants by one-way ANOVA followed by Bonferroni’s multiple comparison test ( ## p < 0.01, ### p < 0.001). ( b , d , f ) Western blotting analysis for phospho-4EBP1 (p-4EBP1). Total cell lysates were prepared 16 h after exposure to 0.2 μg/ml TPG. The levels of depletion of SMG1 and SMG7 using Smg1 and Smg7 siRNAs are shown in Supplementary Fig. f. Cropped blots are shown; all gels were run under the same experimental conditions. Raw data were shown in Supplementary Fig. with dividing lines and with quantitative data.

Article Snippet: Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′) siRNA was designed using the i-Score Designer and asymmetric siRNA was synthesized (GeneDesign, Inc.).

Techniques: Knockdown, Quantitative RT-PCR, Comparison, Western Blot

Journal: Scientific Reports

Article Title: Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

doi: 10.1038/s41598-018-38015-2

Figure Lengend Snippet: Effects of Mtor knockdown on NMD activity ( a , b ), ATF4 expression ( c , d ). ( a ) Analysis of Hnrnpl_NMD mRNA by RT-qPCR. Total RNA was extracted from cells treated 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 shown are the 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 NS siRNA-transfectants by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.001). ( b ) Western blotting analyses of expression of mTOR, p-4EBP1, and NMD components. Total cell lysates prepared 16 h after exposure to 0.2 μg/ml TPG were analyzed using the indicated antibody probes. Cropped blots are shown; all gels were run under the same experimental conditions. Raw data are shown in Supplementary Fig. with dividing lines and with quantitative data. ( c ) Analysis of Atf4 mRNA by RT-qPCR. Total RNA was extracted from cells treated with 0.2 μg/ml TPG for 16 h. The histogram depicts Atf4 mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated NS siRNA-transfectants. Values shown are the 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 NS siRNA-transfectants by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.01). ( d ) Western blotting analyses of stress-related protein expression. Total cell lysates prepared 16 h after exposure to 0.2 μg/ml TPG were analyzed using the indicated antibody probes. Cropped blots are shown; all gels were run under the same experimental conditions. Raw data are shown in Supplementary Fig. with dividing lines and with quantitative data.

Article Snippet: Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′) siRNA was designed using the i-Score Designer and asymmetric siRNA was synthesized (GeneDesign, Inc.).

Techniques: Knockdown, Activity Assay, Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Scientific Reports

Article Title: Environmental stresses suppress nonsense-mediated mRNA decay (NMD) and affect cells by stabilizing NMD-targeted gene expression

doi: 10.1038/s41598-018-38015-2

Figure Lengend Snippet: Effect of mTOR knockdown on expression of membrane transporters. ( a ) Analyses for membrane transporter mRNA expression by RT-qPCR. Total RNA was extracted from cells that were untreated or treated with 0.2 μg/ml TPG for 16 h. The histogram depicts the indicated mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated NS siRNA-transfectants. Values shown are the mean ± SE of four separate experiments. ** , ***Significantly different from TPG-untreated cells by one-way ANOVA followed by Bonferroni’s multiple comparison test (**p < 0.01, ***p < 0.001). ##,### Significantly different from NS siRNA-transfectants by one-way ANOVA followed by Bonferroni’s multiple comparison test ( ## p < 0.01, ### p < 0.001). ( b ) Western blots for membrane transporters in C2C12-DMPK160 cells transfected with NS, mTOR siRNA. Cropped blots are shown; all gels were run under the same experimental conditions. Raw data are shown in Supplementary Fig. with dividing lines and with quantitative data.

Article Snippet: Mouse Eif2α (target sequence: 5′-CAAUGUUGUUAUGUUCU-3′) siRNA was designed using the i-Score Designer and asymmetric siRNA was synthesized (GeneDesign, Inc.).

Techniques: Knockdown, Expressing, Membrane, Quantitative RT-PCR, Comparison, Western Blot, Transfection