KLF8 regulates C/EBPα and PPARγ2 promoters to induce transcription.

<p>(A) A series of C/EBPα promoter constructs cloned into the pGL3-Basic vector was used in this study. NIH3T3 cells were transfected with the luciferase constructs using Lipofectamine, and the luciferase activities were determined after 2 days. The plasmids pcDNA3.0, pCMV-C/EBPβ, or pCMV-KLF8-FLAG were cotransfected. (B) Similarly, the PPARγ2 promoter (−350 counted from transcription initiation site) was investigated by luciferase assay. (C) Mutation analysis of the C/EBPα promoter was performed. The −205 construct was used for the site directed mutagenesis in order to introduce KLF and/or C/EBP regulatory element between −191 and −178 region. The KLF8 binding site was indicated in blue, whereas the C/EBP site was marked in red. The mutated region was underlined. NIH3T3 cells were transfected with these constructs along with KLF8 and/or C/EBPβ overexpression plasmids. (D) Mutation analysis of the PPARγ2 promoter was performed. The KLF site was mutated by site-specific mutagenesis, and the resulting construct was analysed by luciferase assay. Data represent the mean ± SD. **<i>P</i><0.01.