Kinetic analysis of peroxisome proliferators activated receptor γ (PPARγ) promoter demethylation, mRNA expression and cell proliferation. (a, b) The timelapse analysis of PPARγ promoter demethylation and mRNA expression during adipogenesis. Preadipocytes were stimulated to differentiate on day 0 (D0), and harvested every 24 or 48 h until day 8 (D8). The methylation status of the -437 bp, -298 bp and -247 bp CpG sites were determined by restriction endonuclease digestion, and the fraction of the promoter fragments in which all of the three sites were unmethylated is represented (a). The mRNA expression levels were measured by real time reverse transcriptase polymerase chain reaction (RT-PCR) and normalized to the level of β-actin mRNA (b). Individual assessments were repeated three times and the means ± SD are represented, respectively. (c) The increase in cell number by mitotic clonal expansion. The number of differentiating cells was counted at the indicated timepoints, and the cell density was calculated.