Figure 2.

Promoter hypomethylation and overexpression of Nsbp1 in intact uterus of DES- or GEN-treated mice but gene silencing of Nsbp1 in oil-treated mice. Comparison of Nsbp1 promoter methylation and mRNA transcript levels of samples at d 19, 6 months, and 18 months as determined by bisulfite genomic sequencing and real-time RT-PCR, respectively. A–E, Methylation status at each CG site of Nsbp1 promoter was indicated as average methylation (%) from all clones of each sample. Solid diamond, oil control; open square, DES 1 μg/kg · d (DES 1); open triangle, DES 1 mg/kg · d (DES 1K); and cross, GEN 50 mg/kg · d (GEN 50K). F–J, Relative gene expression of Nsbp1 of d-19 samples from oil-treated mice was set to one. Columns, mean; bars, sd; open bar, mice were OVX before puberty; gradient shading bars, OVX mice treated with DES; dot bar, OVX mice treated with GEN; solid bar, mice without ovariectomization (INTACT). Dark, oil control; light gray, DES 1; dark gray, DES 1K; and dot and solid bar, GEN 50K. All data groups were analyzed by ANOVA, followed by post hoc Bonferroni tests with two-tailed distribution, and significant differences between groups were accepted at P < 0.05. *, P < 0.05 when compared with the OVX samples.