Figure 2. In HaCaT cells, RA activates both PPARβ/δ and RAR.
a. and b. Transactivation assays in HaCaT cells transfected with a PPRE-driven luciferase reporter. Cells were treated with the denoted ligands for 15 hr, lysed, and luciferase activity measured and corrected for β-galactosidase activity. Data were normalized to the basal activity. Data are mean ± SEM, n=3 A. Cells were treated with vehicle or GW0742 (GW) or TTNPB (T, 1 μM). B. Cells were cotransfected with either control siRNA or siRNA for PPARβ/δ, and than treated with RA at the denoted concentrations. C. HaCaT cells were treated with denoted ligands (0.1 μM, 4 hr) and RNA extracted. Levels of mRNA of the PPARβ/δ target genes FIAF, ADRP, and PDK-1 were analyzed by Q-PCR and normalized to 18s mRNA. Data are mean ± SEM, n=3. D. HaCaT cells were transfected with control siRNA or PPARβ/δ siRNA (24 hr.), and then treated with the denoted ligands (0.1 μM, 4 hr) and RNA extracted. ADRP mRNA was analyzed by Q-PCR and normalized to 18s mRNA. Data are mean ± SEM, n=3. E. Cells were treated with denoted ligands (0.1 μM, 12 hr) and lysed. Thr-307-phospho-Akt, total Akt, and β-tubulin were assessed by immunoblots. Data from a representative experiment which was repeated 4 times with similar results are shown. F. Transactivation assays in HaCaT cells transfected with a RARE-driven luciferase reporter. Cells were treated with RA at the denoted concentrations (15 hr), lysed, and luciferase activity measured and corrected for β-galactosidase activity. Data were normalized to the basal activity. Data are mean ± SEM, n=3. G. Cells were treated with the denoted ligands and expression of mRNA of the RAR target gene Cyp26a analyzed by Q-PCR and normalized to 18s mRNA. Data are mean ± SEM, n=3.