Activation of peroxisome proliferators activated receptor γ (PPARγ) gene by 5'-aza-cytideine (5'-aza-C) and evaluation of promoter activity with a luciferase assay. (a, b) Expression of PPARγ mRNA in 5'-aza-C-treated NIH/3T3 and 3T3-L1 preadipocytes. The cells were cultured in growth medium containing 5'-aza-C at the indicated concentrations for 48 h before harvest. The mRNA expression levels were determined by real time reverse transcriptase polymerase chain reaction (RT-PCR) and normalized to the levels of β-actin mRNA measured in parallel experiments (n = 3, mean ± SD). The expression level of PPARγ mRNA in differentiated day 6 3T3-L1 adipocytes was also presented at the right of (b) for comparison. (c) Luciferase expression from PPARγ promoter reporter constructs in the presence or absence of in vitro DNA methylation in NIH/3T3, 3T3-L1 preadipocytes and differentiating adipocytes (day 4). Approximately 1 kb of the region upstream of the PPARγ transcription start site (TSS) was cloned into a luciferase reporter vector, and the vector was methylated in vitro as needed. Relative luciferase activity, normalized to the activity of a cotransfected internal control vector, is shown (n = 3, mean ± SD).