Cellular and Biophysical Pipeline for the Screening of Peroxisome Proliferator-Activated Receptor Beta/Delta Agonists: Avoiding False Positives

Figure 2

Optimization of the screening protocols. When not specified, the positive control was 1 μM of GW0742 and the number of cells was 4 × 10⁴ cell/well. Bar graphs represent the specific fold activation normalized by the highest activation presented as the mean ± SD. values were calculated by the unpaired -test ( and ). All bar graphs and calculations were performed with GraphPad Prism. (a) Evaluation of substrate volumes. Cell lysis was performed in 20 μL of passive lysis buffer, and the volumes of LAR II and Stop & Glo substrates were varied (50, 25, and 20 μL). The results show that the best signal of PPARß/δ activation was measured with 25 μL of each substrate. The data is from one experiment with 6 technical replicates. (b) Evaluation of the best ligand incubation medium. In this test, 293T cells were incubated with GW0742 in 10% charcoal-stripped FBS DMEM (charcoal) or in DMEM (incomplete), and the activation fold of PPARß/δ was measured in each condition. -factor charcoal = 0.62; -factor incomplete = 0.21. Charcoal-supplemented medium was chosen as the best option for our assays, and this medium composition was used in the ligand screening for PPARβ/δ. One representative experiment is shown out of three independent replicates. (c) Evaluation of the number of cells per well. Cells were seeded at 2 × 10⁴, 3 × 10⁴, 4 × 10⁴, 5 × 10⁴, and 6 × 10⁴ cells per well, and the activation fold of PPARß/δ was measured in each condition to determine the best quantity of cells in each assay. Based on these results, we chose 4 × 10⁴ cells per well to perform PPARß/δ ligand screening. One representative experiment is shown out of four independent replicates. (d) PPARß/δ dose-response activation in the presence of the commercial agonists (GW0742, GW501516, and L-165,041). Concentrations varied from 10⁻¹¹ to 10⁻⁶M. Data are expressed as the normalized activation fold as 1 (maximum activation) and 0 (vehicle-treated cells) and represent the mean of 2 independent experiments with 3 technical triplicates. Graphs and dose-response calculations were performed in OriginPro 8.0. GW0742: = 0.98549 and EC₅₀ = 1.08708; GW601516: = 0.98028 and EC₅₀ = 7.10385; L-165,041: = 0.9725 and EC₅₀ = 2.23969.

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