Figure 8. Analysis DGAT2 regulation by C/EBPα during adipogenesis.

A, 3T3-L1 preadipocytes were transfected with control siRNA (black bars) or siRNA targeting C/EBPα (white bars) and subsequently induced to differentiate for various times. C/EBPα mRNA expression was assayed by real-time PCR. Data are +/− SEM, normalised to cyclophilin, n=4. B, cells transfected with control (black bars) or C/EBPα (white bars) siRNA were also assayed for DGAT2 mRNA expression by real time PCR. Data are +/− SEM normalised to cyclophilin, n=4. * indicates statistically significant difference in expression compared with control siRNA transfected cells at the same time-point (p<0.05). C, 3T3-L1 preadiocytes were induced to differentiate for various times and subjected to ChIP analysis to assess binding of C/EBPα to the putative site 1 in the DGAT2 promoter. C/EBPα bound DNA in immunoprecipitates was quantified by real-time PCR. Data are the average of 4 independent experiments +/− SEM, * indicates statistically significant difference from binding at time 0 (p<0.05). D, Nuclear extracts were prepared from 3T3-L1 preadiopocytes before or after differentiation for various times as indicated and incubated with biotinylated DNA probe corresponding to site 1 identified in the DGAT2 promoter. Nuclear extracts were incubated with probe alone (−) or with probe in the presence of either anti-C/EBPα antibody (a), or antibody and a 100-fold excess of unlabelled DNA probe (c). Control lanes were included containing probe alone (p) or 24h nuclear extract in the absence of probe (n). S indicates nuclear protein complexes binding probe, and SS indicates antibody-supershifted complexes containing C/EBPα. Data are representative of 3 independent experiments.