Figure 5. NF-κB mediates the response of human osteosarcoma cells to AREG stimulation.

A–C. Cells were pretreated with 0.1% DMSO as control, PDTC (10 μM) or TPCK (10 μM) for 30 min or were transfected with dominant negative gene of IKKα or IKKβ for 24 hr, followed by AREG treatment for 24 hr. The cell migration and ICAM-1 expression were then measured. D. Cells were treated with AREG (50 ng/ml) for the indicated times and the levels of phosphorylated IKKα/β, IκBα, and p65 were examined by Western blotting. E. Cells were pretreated with 0.1% DMSO as control, LY294002 (10 μM), Wortmannin (1 μM) or AKTi (1 μM) for 1 hr followed by stimulation with AREG for 30 min. The level of IKKα/β, IκBα, or p65 and their phosphorylated forms were determined by Western blotting. F. Cells were pretreated with 0.1% DMSO as control, LY294002 (10 μM), Wortmannin (1 μM) or AKTi (1 μM) for 30 min, followed by AREG treatment (50 ng/ml) for 120 min. Chromatin immunoprecipitation was performed with antibody against p65. One percent of immunoprecipitated chromatin was assayed to verify equal loading (Input). G–H. Osteosarcoma cells were transfected with a plasmid harboring the NF-κB luciferase reporter for 24 hr and then the stable cell lines were treated with indicated inhibitors for 30 min or was transfected with different dominant negative genes for 24 hr. Luciferase activity was measured after AREG stimulation for 24 hr. Data are shown as the mean ± SEM. The asterisks indicate t-test comparisons to the control of 0.1% DMSO treatment or to cells transfected with the empty vector (Vector). ***represents P < 0.001. #represents P < 0.05 for one-way ANOVA comparisons as indicated. ##represents P < 0.01. ###represents P < 0.001.