2.7. Transient Transfection and Dual Luciferase Reporter Assay

After reaching 70–80% confluency, cells were transiently transfected with 50 ng of a 3X ACO-PPRE vector (containing three copies of consensus PPRE from the ACO promoter in front of a luciferase reporter gene) and pGL4.10 vector as negative control using FuGENE 6 transfection reagent (Roche diagnostics, Mannheim, Germany) for 12 h according to the manufacturer’s protocol. Cells were also co-transfected with 5 ng of pGL4.74 Renilla luciferase (encoding the Renilla luciferase reporter gene; Promega, Mannheim, Germany), which was used as internal control reporter vector to normalize for differences in transfection efficiency (Promega). After transfection, cells were treated with either WY-14643; the isolated fatty acids LA, ALA, MTriA, MTA, IPA or MHD; or the CHLE at the concentrations indicated or vehicle alone for 24 h. Afterwards, cells were washed with PBS and lysed with lysis buffer (Promega). Luciferase activities were determined with Beetle-Juice and Renilla-Juice Kits from PJK (Kleinblittersdorf, Germany) in a Mithras LB940 luminometer (Berthold Technologies, Bad Wildbad, Germany). For control of background luminescence, Firefly and Renilla luciferase activities were also determined in the lysates of nontransfected control cells and subtracted from luminescence of transfected cells. Data were normalized for transfection efficiency by dividing Firefly luciferase activity of the 3X ACO-PPRE plasmid by that of Renilla luciferase activity of the co-transfected pGL4.74 Renilla luciferase plasmid. Results represent normalized luciferase activities and are shown relative to cells transfected with pGL4.10 vector and treated with the vehicle only, which was set to 1.