PPAR transactivation assays and EC50 determination

A one‐hybrid‐system, using hybrid constructs of GAL4 DNA‐binding part coupled to PPARα, PPARβ or PPARγ ligand binding domains together with a luciferase reporter and a renilla‐expressing plasmid, was applied. For transient transfections, white 96‐well cell culture plates with clear bottom (Corning, Basel, Switzerland) were used. As many as 7.5 × 10⁴ HEK293 cells per well were plated in minimum essential medium (Eagle) at 37°C in 5% CO₂ without phenol red supplemented with 10% charcoal‐treated fetal bovine serum (HyClone Laboratories, Inc., Logan, UT, USA). The cells were transiently transfected at 70–80% confluence by polyethylene‐mine‐based transfection for 5 h at 37°C, 5% CO₂, which was followed with respective stimulations of the applied compounds dissolved in DMSO (0.45% final DMSO concentration in the wells). The GW7647 compound was used as a reference for human PPARα. Stimulations lasted 16 h according to established protocols. Transfection efficiency was adjusted to renilla expression (Promega AG, Dübendorf, Switzerland). All concentrations were tested in three biological replicates. The ‘dose response for one site’ was applied in a curve‐fitting model according to the formula: y = A + ((B − A)/(1 + ((10C)/x)D)), where A is the minimum y‐value, B the maximum y‐value, C Log EC50 and D the slope factor. The data were fitted by XLfit (http://www.idbs.com) using the Levenberg Marquardt algorithm.