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. 2017 Aug 11;7:7927. doi: 10.1038/s41598-017-08513-w

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© The Author(s) 2017

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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2-ME induced insulin secretion in MIN6 cells. (a) Time-dependent insulin secretion by 2-ME (5, 50, 100 and 500 nM concentrations) at 3 mM, 6 mM and 30 mM medium glucose concentrations. Insulin estimation by the ELISA method performed in triplicate. Three sets of independent experiments were performed. (b,c) Western blot data analysis of control and 2-ME-treated MIN6 cells. A representative image of 5 blots is shown. Cropped images were displayed and original blots are shown in the figure Supplementary 14. Densitometry data were normalized to β-actin. (d) Time-dependent insulin secretion by 2-ME in the scramble and AMPK siRNA transfected MIN6 cells. Scramble and AMPK siRNA were transfected using lipofectamine 2000 at 100 nM concentration in cells. Insulin estimation was performed in triplicate. Three sets of independent experiments were analyzed. (e) Western blot analysis of total AMPK and AMPK phosphorylation protein levels in the scramble and AMPK siRNA transfected MIN6 cells. A representative image from 5 blots is shown. Cropped images were displayed and original blots are shown in the figure Supplementary 14. Densitometry data normalized to β-actin. (f) Western blot analysis of total AMPK and AMPK phosphorylation after treatment with 2-ME and AICAR at 3 mM glucose and 30 mM glucose concentration. A representative image from 4 blots is shown. Cropped images were displayed and original blots are shown in the figure Supplementary 15. (g) Time-dependent insulin level in 2-ME and AICAR-treated cells in the 3 mM glucose and 30 mM glucose media concentration. Insulin estimation assays were performed in triplicate. (h,i) Western blot analysis of PDX1, PDX1 phosphorylation and MST1 in 2-ME and AICAR-treated cells under 3 mM and 30 mM of glucose concentration for 24 hours. A representative image of 4 blots is shown. Cropped images were displayed and original blots are shown in the figure Supplementary 16. Densitometry data were normalized to Actin. N = 4 were analyzed in each data set. The data in the graph are shown as the mean ± s.e.m. Prism3 software was utilized for the statistical calculation. The One way Anova (Tukey test) was carried out to determine of statistical significance.