Figure 3

Effects of PPAR agonists on iNOS expression in activated J774 macrophages. (a) Effects of peroxisome proliferator-activated receptor (PPAR) agonists on inducible nitric oxide synthase (iNOS) mRNA expression. Cells were treated with PPAR agonists, NF-κB inhibitor MG132 or vehicle (dimethyl sulfoxide, DMSO) and stimulated with LPS for six hours. Total RNA was extracted and iNOS mRNA was determined by RT-PCR. The results were normalized against GAPDH mRNA. (b) Effects of PPAR agonists on iNOS mRNA degradation. Cells were treated with PPAR agonists or vehicle (DMSO) and stimulated with lipopolysaccharide (LPS). After six hours incubation, actinomycin D (1 µg/ml) was added to the cell culture to stop transcription. Total RNA was extracted at indicated time points after actinomycin D addition, and iNOS mRNA was determined by RT-PCR. The results were normalized against GAPDH mRNA. (c) Effects of PPAR agonists on iNOS promoter activity in J774 macrophages stably transfected with a luciferase (LUC) reporter gene under the control of iNOS protein. Cells were treated with PPAR agonists, NF-κB inhibitor MG132 or vehicle (DMSO) and stimulated with LPS for six hours. Total RNA was extracted and LUC mRNA was determined by RT-PCR. The results were normalized against GAPDH mRNA. (d) Post-transcriptional effects of PPAR agonists on iNOS protein levels. Cells were stimulated with LPS for 10 hours. After the stimulation, the culture medium was changed and the cells were further incubated with PPAR agonists or vehicle (DMSO) without LPS for additional 14 hours. Then, proteins were extracted and the levels of iNOS protein were analysed by Western blotting. β-actin was used as a loading control. Results represent the mean + SEM (n = 3 to 4). In d one representative Western blot is shown. *P <0.05 and **P <0.01 as compared to cells treated with LPS alone.