Effects of PPARα and δ agonists on the ICAM-1 promoter activity and mRNA half-life. a Analyses of wildtype ICAM-1 luciferase (Luc) reporter construct in HUVECs. The Luc activity is expressed in percent of control (mean ± SEM of three independent experiments each performed in triplicate). HUVECs were left untreated (solvent only) or were treated with different concentrations of PPARα agonsist (WY14643 (200 μM) and PPARδ agonists (L-165041 (50 μM)) for 24 h. As positive control TNFα (20 ng/ml) was used. Mean values from triplicate experiments with four independent experiments are depicted ± SEM. *p < 0.05 was considered significant. b Analyses of 5′-deletional ICAM-1 promoter-based luciferase constructs in HUVECs. Schematic representation of the respective reporter gene constructs on the left and the relative Luc activities (expressed as % activity of the control cells) in graphic format on the right. HUVECs were left untreated (solvent only) or were treated with the PPARδ agonists (L-165041 (50 μM)) for 24 h. Results were confirmed in four independent sets of experiments. *p < 0.05.
c Representative EMSAs using nuclear extracts of HUVECs that were left untreated (solvent only) or were treated with PPARδ agonists L-165041 (50 μM) for 24 h (lane 1,2): mutated labelled Sp1 DNA (lane 3), competition with unlabelled wild-type DNA (lane 4, at 100 molar excess) or with unlabelled excess double-stranded Sp1 consensus oligonucleotides (lane 6, at a final concentration of 0.35 lmol ⁄ l). Supershift analyses were performed by addition of specific Sp1 (lane 7 and 8) or Sp3 antibody (lanes 9 and 10, all from Santa Cruz)) at a final concentration of 100 ng ⁄ ml. Formation of Sp-dependent binding complexes is indicated by arrows to the left. A representative autoradiography from three independent experiments is shown. d HUVEC were incubated with vehicle, L165041 (50 μM) or WY14643 (200 μM) for 1 h, followed by incubation with fresh media containing Act D (10 μg/ml) for 0, 12, 24 and 36 h. RT-PCR analyses for ICAM-1/GAPDH of total RNA extracted from subconfluent cell cultures were performed. The PCR products were separated by 2 % agarose gel electrophoresis, and ethidium bromide stained bands visualized using an ultraviolet transilluminator. ICAM-1 bands were quantified by densitometric scanning, the results of which were normalized to amounts of GAPDH mRNA. The mean values from five independent experiments are presented as the mean ± SEM. *p < 0.05