FIG. 9.

Transfection with siRNA against p38 inhibits the activation of IKK and NF-κB and the expression of hBD-2 in HT-29 cells stimulated with BFT. (A and B) The siRNA-transfected cells were cotransfected with p2x NF-κB- or wild-type hBD-2-luciferase reporter for another 24 h. BFT (300 ng/ml) was added to cotransfected cells for 1 h (NF-κB) (A) or 6 h (hBD-2) (B). Data are expressed as mean fold induction ± SEM of luciferase activity relative to untreated controls (n = 5). The mean fold induction of the β-actin reporter gene relative to untreated controls remained relatively constant throughout each experiment. Asterisks indicate values of BFT plus siRNA that are significantly different from those of BFT alone (P < 0.05). (C) BFT (300 ng/ml) was added to the siRNA-transfected HT-29 cells for the indicated period. IKK kinase activity was measured using an HTScan IKK-β kinase assay kit. Data are expressed as mean fold induction ± SEM of kinase activity relative to untreated controls (n = 5). Asterisks indicate values of BFT plus siRNA that are significantly different from those of BFT alone (P < 0.05). (D) The siRNA-transfected cells were combined with BFT (300 ng/ml) for 30 min. Cell lysates were analyzed by immunoblotting with the indicated antibodies. Results shown are representative of three independent experiments. Lanes: 1, unstimulated control in nontransfected cells; 2, BFT in nontransfected cells; 3, BFT in p38 siRNA-transfected cells; 4, BFT in NS-RNA-transfected cells.