Figure 8

The combination of GGTI-298 and TRAIL augments Akt inhibition, IκBα degradation ( A ) and NF-κB activation ( B ), which are differentially modulated by DR4 and DR5 ( C ). A, A549 cells were treated with the combination of 15 μM GGTI-298 and 20 ng/ml TRAIL for the given times (upper panel) or with DMSO (D), 15 μM GGTI-298 (G) alone, 20 ng/ml TRAIL (T) alone or GGTI-298 and TRAIL combination (G/T) for 2 h (lower panel). The cells were then subjected to preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blotting. B, A549 cells stably transfected with NF-κB-luc reporter gene were treated with DMSO, 15 μM GGTI-298 alone, 20 ng/ml TRAIL alone or GGTI-298 and TRAIL combination for 3 h. The cells were then lysed for NF-κB luciferase assay, which was finally normalized by protein content. The columns are means of triplicate determinations; Bars, ± SDs. C, A549 cells were cultured in 6-well plates and the next day transfected with the indicated siRNAs twice in a 48 h interval. Twenty-four hours after the second transfection, the cells were treated with 15 μM GGTI-298 plus 20 ng/ml TRAIL (G/T) for the indicated time and then subjected to preparation of whole-cell protein lysates and subsequent detection of the given proteins using Western blotting.