(A) Representative pancreatic sections from 2-week-old 3aRCY-KO and littermate-control 3aRCY-Het animals, immunostained for insulin (Ins; green) and glucagon (Glu; red), with DAPI (blue) (left 2 panels), or NKX6.1 (red), insulin (Ins; green) and DAPI (blue) (right 2 panels). Scale bar: 100 μm. (B) Relative mRNA expression of indicated genes in sorted β cells from 6-week-old 3aRCY-KO and control 3aRCY-Het littermates. Cyclophilin A was used as a housekeeping gene. (C) Bisulfite sequencing analysis for the Hk1 and Ldha loci at indicated regions comparing sorted β cells from 6-week-old 3aRCY-KO and control 3aRCY-Het littermates (representative clones from n = 3 mice). Each horizontal line with dots is an independent clone, and 10 clones are shown here. These regions are almost fully DNA-methylated (filled circles) in β cells from 3aRCY-Het mice, but largely hypomethylated (open circles) in β cells from 3aRCY-KO mice. For all experiments, n = 3 independent experiments. The error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test.