PPAR-γ promoter cloning and reporter gene assay

The detailed method for the DNA cloning experiments was described in a previous report³⁵. The 1.0 kb DNA fragment harboring the mouse PPAR-γ proximal promoter was amplified from mouse genomic DNA using PCR. The fragment was then subcloned into a pGL3-Basic vector. The construct was confirmed by DNA sequencing. To obtain the unmethylated promoter, the reporter constructs were transformed into the dam-/dcm- E. coli strain (New England Biolabs, Ipswich, MA, USA). To obtain the fully methylated reporter, constructs were incubated with 3 U/μg of SssI methylase (New England Biolabs) in the presence of 160 μM S-adenosylmethionine at 37 °C for 3 hours³⁶. The transfection was performed following the protocol of Lipofectamine-2000 (#12566014, Invitrogen). The luciferase activities of the cell lysates were evaluated according to the manufacturer’s instruction (#E1910, Promega), and the total protein concentration in each assay was measured as an internal control.