Effects of PPAR agonists on NF-κB activation and NF-κB-mediated transcription. (a) Effects of peroxisome proliferator-activated receptor (PPAR) agonists on NF-κB translocation to nucleus. J774 cells were preincubated with PPAR agonists or vehicle (dimethyl sulfoxide, DMSO) for two hours before addition of lipopolysaccharide (LPS). After 30 minutes incubation with LPS, nuclear extracts were prepared. The levels of NF-κB p65 in nuclear extracts were analysed by Western blotting. Lamin A/C was used as a loading control. (b) Effects of PPAR agonists on DNA binding activity of NF-κB. J774 cells were preincubated with PPAR agonists, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) or vehicle (DMSO) for two hours before addition of LPS. After one hour incubation with LPS, nuclear extracts were prepared and the binding activity of NF-κB p65 to DNA was analysed as described in methods. (c) Effects of PPAR agonists on NF-κB-mediated transcription activity. HEK293 cells were stably transfected with a luciferase (LUC) reporter gene under the control of NF-κB-responsive promoter. The cells were preincubated with PPAR agonists, NF-κB inhibitor PDTC or vehicle (DMSO) for two hours before addition of TNFα. After six hours' incubation with TNFα, total RNA was extracted and LUC mRNA was determined by RT-PCR. The LUC mRNA levels were normalized against GAPDH mRNA. Results represent the mean + SEM (n = 3 to 4). In a one representative Western blot is shown. **P <0.01 as compared to cells treated with LPS or TNFα alone.