Fig 1.
Analysis of the proliferation potency of MIN6 cells at different passage frequencies.
(A) Proliferation data comparing Lpn (dotted line) and Hpn (straight line) MIN6 cells. (B, D) Immunoblot analysis of PCNA (B) and insulin signaling proteins (D) in Lpn and Hpn MIN6 cells. Representative (left) and quantitative (right) data are shown. (C) Representative cell cycle analysis in Lpn (left) and Hpn (right) MIN6 cells. (E) Quantitative real-time PCR analysis of insulin signaling molecule mRNA expression in Lpn (white bars) and Hpn (black bars) MIN6 cells. (F) Immunoblot analysis of IRS2 in Lpn and Hpn MIN6 cells. Representative (left) and quantitative (right) data are shown. (G, H) Representative (left) and quantitative (right) immunoblot analysis of insulin signaling proteins (G) and representative cell cycle analysis (H) in Hpn cells with or without LY294002. Data are represented as the mean ± SEM for 4 (A), and 5 (B–G) independent experiments. *P < 0.05.
Fig 2.
Analysis of the epigenetic regulation of insulin signaling molecules in pancreatic β cells.
(A) Immunoblot analysis of the transcription factor CREB in Lpn and Hpn MIN6 cells. Representative (left) and quantitative (right) data are shown. (B) Combined bisulfite restriction analysis of DNA methylation. (C) Bisulfite sequence of the Irs2 promoter region in Lpn and Hpn MIN6 cells. Black circles indicate methylated CpG sites and white circles indicate non-methylated CpG sites. Percentage is the ratio of methylated CpG sites. (D) Representative ChIP analysis of H3K9/14 histone acetylation of the Irs2 promoter region in Lpn and Hpn MIN6 cells. (E) ChIP-qPCR of H3K9/14 histone acetylation of the Irs2 promoter region in Lpn and Hpn MIN6 cells. (F) ChIP analysis of H3K9/14 histone acetylation of the Irs2 promoter region and immunoblot analysis of IRS2 in islets isolated from db/db or control db/m mice at 10 weeks of age. Representative (left) and quantitative (right) data are shown. Data are represented as the mean ± SEM for 5 (A–F) independent experiments. *P < 0.05.
Fig 3.
Effects of specific HDAC inhibitors on the expression of IRS2 in MIN6 cells.
(A–C) Immunoblot analysis of IRS2 in MIN6 cells treated with the DNA methylation inhibitor 5-aza-dC (A) or the class I and class II HDAC inhibitors TSA (B) and SAHA (C). Representative (left) and quantitative (right) data are shown. (D, E) Quantitative real-time PCR analysis of Irs2 mRNA expression in control MIN6 cells (white bars) and those treated with TSA (D) or SAHA (E) (black bars). (F, G) ChIP-qPCR of H3K9/14 histone acetylation of the Irs2 promoter region in MIN6 cells with TSA (F) or SAHA (G). (H) Quantitative real-time PCR analysis of HDAC isoforms in Lpn and Hpn MIN6 cells. Data are represented as the mean ± SEM for 5 (A–H) independent experiments. *P < 0.05.
Fig 4.
Effects of specific HDAC inhibitors on insulin signaling through IRS2 lysine acetylation in MIN6 cells.
(A) Immunoblot analysis of insulin signaling proteins in MIN6 cells treated with TSA or SAHA. Representative (left) and quantitative (right) data are shown. (B) Immunoblot analysis of IRS2, acetylated lysine, and phosphorylated tyrosine in MIN6 cells treated with TSA after protein interaction analysis using antibodies to IRS2. Representative (left) and quantitative (right) data are shown. (C) Proposed model of HDAC regulation of IRS2 expression and activity in β cells. Data are represented as the mean ± SEM for 5 (A, B) independent experiments. *P < 0.05.