Figure 5

AP-1 and NF-κB transcription factors are involved in sulindac-sulfide induced activation of IL-8 gene expression. (A) HCT-15 cells were treated with 50 μM sulindac sulfide (SS) or the control DMSO for 4 hours. Western blot analysis of the cytoplasmic and nuclear fraction for phosphorylated c-Jun (and phosphorylated junD), c-Jun, c-Fos and p65. PARP is used as a nuclear marker while β-tubulin is used as a cytoplasmic marker. Loading control β-actin. Representative blot of two independent experiments. (B) qPCR analysis for c-JUN and c-FOS. Bars represent fold change of relative gene expression, normalized to the house-keeping gene GAPDH. Mean values ± SEM (from three independent experiments). (C) Luciferase reporter gene assay was performed to measure transcriptional activity from the human endogenous IL-8 promoter. HCT-15 cells were transfected with the IL-8 promoter reporter (-133-luc) or reporters with mutated AP-1 and NF-κB binding sites (-133(AP-1-mut)-luc and -133(NF-κB-mut)-luc respectively) and β-glactosidase reporter and treated with 20 μM sulindac sulfide (SS), the vehicle control DMSO (control) or the positive control TNFα (20 ng/ml) for 2 hours. Luciferase activity was normalized to β-galactosidase activity and is presented as mean Relative Luciferase Activity ± SEM (from three independent experiments performed in duplicate). * represents P < 0.05.