Fig. 6

EZH2 inhibitors influence glucose-sensitive insulin secretion in human pancreatic ductal cells. A Gene expression of chromatin modulators EZH2, P300, KAT2 and BRG1 are unchanged in non-inhibitor-treated cells following exposure to high glucose (HG) compared to non-exposed (LG) cells. Data are presented as mean of fold change ± S.E.M. of 2 replicates, calculated by normalizing high glucose values to low glucose (unexposed) controls. Statistically significant change in expression was determined using Student’s t-test. B Representative western blots and quantitative analysis of EZH2 and H3K27me3 following exposure to high glucose (HG) compared to low glucose (LG). Data are displayed as mean signal ratio of EZH2 to β-actin or H3K27me3 to total H3 ± SEM of 2 replicates with representative blots above. Each dot plot represents signal ratio of one independent replicate. Statistically significant differences were determined using Student’s t-tests against control. C 2- and 7-day protocols for assessment of glucose-stimulated insulin secretion from EZH2 inhibitor (EZH2i) treated human pancreatic ductal epithelial cells. Both protocols were initiated with seeding of cells to establish cultures. Two-day EZH2i stimulation was performed in CMRL to resolve background insulin, whilst for 7-day stimulations, the initial EZH2i doses were delivered in normal growth media, followed by switching to CMRL on day 6. On the final day of the protocol, cells were incubated for 1 h in low glucose followed by 1 h in high glucose. The supernatant was collected for quantification of insulin secretion in ELISAs. ELISA quantified D 2- and E 7-day secretion of insulin from human pancreatic ductal epithelial cells following 1 h of incubation in low (2.8 mM) and high (28 mM) concentrations of glucose. Insulin concentrations were normalized to control 2.8 mM concentrations to calculate fold change. Data are presented as mean of fold change ± S.E.M. of 3 replicates. Dots represent one technical replicate of 2.8 mM glucose supernatant. Triangles represent one technical replicate of 28 mM glucose supernatant. Student’s t-tests were used to assess whether variation in insulin secretion was statistically significant, *P < 0.05, **P < 0.01, ****P < 0.0001