Cytokine expression in islets and their effects on insulin DNA methylation. (a) Cytokine gene transcription in islets from NOD mice was determined by qRT-PCR and presented as fold changes relative to levels in 3-week-old NOD mice. (b, c) Islets from 4-week-old NOD mice were cultured in medium without (white bars) or with either IL-6 plus IL-1β (horizontal stripes) or IFN-γ plus IL-1β (diagonal stripes) for 48 h. (b) Insulin gene transcription was analysed in sorted beta cells by qRT-PCR and normalised to transcription levels in beta cells from islets cultured in medium alone. (c) Methylation status of Ins1 was determined by cloning and sequencing Ins1 exon 2 (χ2 test, p<0.0001). Data are the means ± SEM of 3–4 experiments, with (a) 3–4 mice per time point or (b, c) 6 mice per group. (a, b) ANOVA: *p<0.01, **p<0.01, ***p<0.001, ****p<0.0001