Figure 2.

Dnmt3a is upregulated with increasing promoter methylation of adipogenic transcription factors during the stage of CI. (a) The mRNA expression of Dnmt3a during the process of 3T3-L1 preadipocyte differentiation. The mRNA level was determined by real-time PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. 'cyc' indicates the stage of cycling cells, 'ci' indicates the CI stage, 'mdi' indicates the stage of differentiation induction by hormone cocktail. Results were expressed as means±S.D. (n=3). Statistical significance is indicated: **P<0.01, ***P<0.001, NS=non-significant. (b) The protein expression of Dnmt3a was detected at indicated time points as described in (a) (left panel). Cyclin-dependent kinase 4 was used as the loading control. Relative quantification analysis of band densities was shown on the right panel. Data were presented as means±S.D. (n=3). Statistical significance is indicated: **P<0.01, ***P<0.001, NS=non-significant. (c) For promoter methylation analysis of adipogenic transcription factors, CpG island analysis of the 5' flanking and 5' untranslated sequences of murine C/EBPβ, C/EBPα, PPARγ and Egr2 are shown. The red vertical line stands for a CpG site. The region indicated by arrows stands for the sequence for bisulfite sequencing. (d) Bisulfite sequencing results of murine C/EBPβ, C/EBPα, PPARγ and Egr2 from 3T3-L1 preadipocytes under the indicated conditions. Each column stands for a CpG site. The methylation percentage of each CpG site was shown by blue in each column. And the non-methylation percentage of each CpG site was shown by yellow in each column. Data were presented as mean from three independent samples