Figure 2
PPARγ Trans-Represses IRF6 in Cerebrovascular Endothelial Cells. (A) Diagram of the pGL 4.10 luciferase reporter plasmid carrying the murine IRF6 gene promoter sequence 5′-upstream of Luc. Two putative PPRE binding sites were identified at −2206/−2190 bp (PPRE site 1) and −11626/−11606 bp (PPRE site 2). Transcription starts at +1 bp. (B) A luciferase reporter assay was conducted by transfecting murine cerebrovascular endothelial cells with the pGL 4.10 luciferase reporter plasmid delivering either the IRF6 wild-type promoter sequence (IRF6 PPRE WT) or the IRF6 promoter sequence carrying a mutation at one of the two PPRE sites (IRF6 PPRE site 1 mutant or IRF6 PPRE site 2 mutant). Pioglitazone and/or PPARγ gain-of-function significantly elevated IRF6 wild-type promoter activity but did not affect IRF6 PPRE site 1 mutant promoter activity. *P < 0.05 versus control group, † P < 0.05 versus Pioglitazone group, ‡ P < 0.05 versus Ad.PPARγ group. (C,D) Real-time PCR on the total RNA content from control and small-hairpin PPARγ RNA (PPARγ shRNA)-transfected murine cerebrovascular endothelial cells revealed that PPARγ silencing significantly elevates IRF6 mRNA expression, particularly more significantly under oxygen-glucose deprivation (OGD) conditions. Cropped blots are displayed here. *P < 0.05 versus same-condition Control group, † P < 0.05 versus Non-OGD PPARγ shRNA group. Data are reported as means ± standard errors of the mean (SEMs).
