Fig. 5.

GSK126 influences the refractory H3K27me3 content on the insulin chromatin domain in human non-diabetic exocrine cells. a Flow chart of EZH2 acid extraction from human pancreatic ductal cells. Purification method of acid histone-associated protein fraction isolated from heterochromatin isolates derived from pancreatic ductal nuclei. Extraction of histone-binding EZH2 protein fraction from heterochromatin involve acid homogenisation and precipitation (ppt) with 5 M H₂SO₄. Isolated histone-binding proteins with EZH2 were fractionated and quantified using Li-COR Odyssey. b Dose-dependent increase of GSK126 (5 or 10 µM) for 48 h attenuates EZH2 in human pancreatic ductal cells. Quantification levels of EZH2 were calculated and adjusted to unmodified histone H3 using Li-COR Odyssey. The signal ratio was calculated as EZH2/overall H3. Vehicle control was DMSO. Ordinary one-way ANOVA was performed on Control vs GSK-126 (*P < 0.05, **P < 0.01, error bars are SEM, n = 3). c Insulin domain was assessed using amplimers that were specifically designed to distinguish promoter regions (R) of the Ins and Igf2AS genes. d Quantitative PCR analysis of DNA in chromatin immunoprecipitated (ChIP) with anti-H3K27me3 antibody. Vehicle control was DMSO. e DNA was assessed using amplimers that specifically recognise the promoter regions (R) of the Ngn3 and Pdx1 genes. f Quantitative PCR analysis of DNA in ChIP with anti-H3K27me3 antibody. Vehicle control was DMSO. Data represented as the mean Input signal against specific H3K27me3 abundance. Student’s t-test was performed on Control vs GSK-126 (*P < 0.05, **P < 0.01 error bars are SEM, n = 2)