Figure 3: High glucose treatment inhibits Oct4 and Nanog expression via APEX1-mediated DNA methylation.

(A,B) Western blot (A) and real-time PCR (B) analyses of OCT4 and NANOG expression in the cervical loops of offspring of control and diabetic dams; *P < 0.05 vs. control; (C) Sodium bisulphite sequencing analyses of DNA methylation levels of the Oct4 promoter (from −533 to −34) and Nanog promoter (from −672 to −332) in the cervical loops of offspring from control and diabetic dams. (D) High glucose treatment decreased the expression of Oct4 and Nanog in dental epithelial stem cells (DESCs), and overexpression of APEX1^WT, but not APEX1^C65A, reversed the downregulation of Oct4 and Nanog induced by high glucose treatment; *P < 0.05 vs. control; ^#P < 0.05 vs. high glucose. (E) Real-time PCR showed decreased expression of Oct4 and Nanog in response to high glucose treatment, Apex1 knockdown, or inhibition of APEX1 redox function by E3330 in primary DESCs; treatment with the DNMT inhibitor 5-aza-dC significantly reversed the downregulation of Oct4 and Nanog; *P < 0.05 vs. control; ^#P < 0.05 vs. HG; ^§P < 0.05 vs. Si-Apex1; ^※P < 0.05 vs. E3330. (F) Sodium bisulphite sequencing showed an increase in Oct4 and Nanog promoter DNA methylation levels in DESCs treated with high glucose, APEX1-specific siRNA or E3330. Overexpression of APEX1^WT, but not APEX1^C65A, decreased DNA methylation levels of the Oct4 and Nanog promoters.