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. 2019 Feb 4;9:1279. doi: 10.1038/s41598-018-38015-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of MeHg or mild ER stress on NMD activity in C2C12-DMPK160 cells (a–c), CNCs (d,e), and AGCs (f,g). (a) RT-qPCR analysis of Snhg1 mRNA. The histogram depicts Snhg1 mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated controls. Values represent mean ± SE of three separate experiments. ***Significantly different from MeHg-untreated cells by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.001). (b) Effects of mild ER stress on NMD activity. C2C12-DMPK160 cells pretreated with TPG (0.2 μg/ml) for 16 h were exposed to 0.5 μM MeHg for 5 or 7 h. The histogram depicts Snhg1 mRNA normalized to Actb mRNA presented as the fold-increase over non-pretreated MeHg-untreated controls. Values represent mean ± SE (n = 3). ***Significantly different from TPG-untreated cells by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.001). (c) Western blotting analyses of NMD components’ protein expression. Total cell lysates prepared 7 h after exposure to 0.5 or 0.8 μM MeHg 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. 1c with dividing lines and with quantitative data. (d,f) RT-qPCR analysis of Gas5 mRNA. The histogram depicts Gas5 mRNA normalized to Actb mRNA represented as the fold-increase over MeHg-untreated controls. Values shown are the mean ± SE of three separate experiments. **^,***Significantly different from MeHg-untreated cells by one-way ANOVA followed by Bonferroni’s multiple comparison test (**p < 0.01, ***p < 0.001). (e,g) Western blotting analyses of expression of NMD component proteins. Total cell lysates prepared 24 h for CNCs or 9 h for AGCs after exposure to the indicated concentration of MeHg 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. 1e, g with dividing lines and with quantitative data.