Ppre x3 tk luc

To investigate the transcriptional activity of PPARs, we used a peroxisome proliferator response element (PPRE)-driven luciferase reporter gene approach (Kim et al., 1998 (link)). In brief, CHO cell lines were transfected with a reporter construct containing three copies of the PPRE placed upstream of the thymidine kinase promoter- firefly luciferase fusion gene (PPRE X3-TK-luc, Addgene plasmid #1015). In addition, the cells were transfected with a cytomegalovirus promoter-driven Renilla luciferase reporter gene (pGL4.75 hRluc/CMV, Promega, E6931) used as transfection control. Where indicated, transfectants were treated with 5 µM of rosiglitazone (Sigma, R2408) or with conditioned medium containing 5 µg/ml of human apoE3 or apoE4 for 24 h. At 24 to 48 h after transfection, cells were lysed in buffer (50 mM Tris-HCl, pH 7.4, 140 mM NaCl and 1% Triton X-100) and lysates used to assay luciferase activity in 96-well plates using the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer's instructions. The luminescence-based PPAR activity (firefly luciferase) was normalized to the internal Renilla luciferase activity.

The fragments encoding CYP1B1 and KLF11 were cloned into the pcDNA3.1 (+) vector. The fragment encoding PPARγ was cloned into the pSG5 vector. The luciferase reporter PPAR response element (PPRE) X3‐TK‐Luc was obtained from Addgene (Watertown, MA). All the constructs were verified by DNA‐sequence analysis. To investigate the regulation of tight junctions by CYP1B1, hCMEC/D3 cells were transiently transfected with CYP1B1 or PPARγ expression vector for 24 h. To investigate the regulation of MAO‐B by CYP1B1, SH‐SY5Y cells were transiently transfected with the CYP1B1 or KLF11 expression vector for 24 h. The respective empty vectors were used as the control.
To investigate the effects of HETEs on PPARγ transcriptional activity, hCMEC/D3 cells were treated with 5‐HETE (1 μM) and 15‐HETE (1 μM) following co‐transfection with PPRE luciferase reporter and PPARγ expression vector for 24 h. The luciferase activity was determined using the Dual‐Luciferase Reporter Assay kit (Promega, Madison, WI, USA), and the firefly luciferase activity was normalized to the Renilla luciferase activity.