Figure 7

SH122 suppressed TRAIL-induced NF-κB activation. A. DU145 cells were treated with 300 ng/ml of TRAIL for the indicated time. Cytosol and nuclei subcompartments were fractionated for detection of IκBα and RelA, with Actin and PARP used as markers of cytosolic and nuclear extracts, respectively. Relative expression of IκBα and RelA was shown by dividing band intensity by that of Actin and PARP, respectively. CE, cytosolic extract; NE, nuclear extract. B. After treatment with 300 ng/ml of TRAIL, cells were harvested for quantitative RT-PCR detect NF-κB target genes. Fold increase of gene expression was calculated by dividing the normalized gene expression activity by that of the untreated control. C. Cells were pre-treated with desired compounds for 1 h, and challenged with TRAIL (300 ng/ml) for 40 min. Cytosol and nuclei subcellular compartments were fractionated for detection of IκBα and RelA, respectively. MG132 was used as a positive control for blocking NF-κB. D. After treatment as described in C, cells were harvested for quantitative RT-PCR to detect three NF-κB target genes. E, Time-course of TRAIL-induced NF-κB activation examined by NF-κB luciferase reporter assay. DU145 cells were transiently co-transfected with pNF-κB or pControl together with β-galactosidase plasmid, and then treated with TRAIL for the indicated time. Luciferase and β-galactosidase activities were measured as described in Materials and Methods. F, SH122 inhibited TRAIL-induced NF-κB activation in NF-κB luciferase reporter assay. Transfected DU145 cells were pretreated with SH122 or SH123 for 1 h followed by TRAIL treatment for 4 h. Fold of NF-κB activation was calculated by dividing the normalized luciferase activity by that of the untreated control. Representative results of at least two independent experiments. Columns, mean; bars, SD (n = 3). Con, DMSO vehicle control. ** P < 0.01; * P < 0.05, Student's t-test (n = 3).