AEG-1 and NF-κB pathway. A. AEG-1 activates the NF-κB pathway. Upper panel, HeLa cells were infected with Ad.AEG-1 (50 pfu/cell) or treated with TNF-[unk] (10 ng/ml) and 2 days later NF-κB binding activity was analyzed in the nuclear extracts of the cells by EMSA. Supershift analysis was performed with the indicated antibodies. *indicates a supershifted band by anti-p50 antibody; ** indicates a supershifted band by anti-p65 antibody. Lower panel, Ad.AEG-1 infection activates NF-κB by IκB[unk] degradation. HeLa cells were infected with Ad.AEG-1 (50 pfu/cell) or treated with TNF-[unk](10 ng/ml). The expressions of the indicated proteins were analyzed in cytoplasmic extracts by Western blot analysis 2 days later. B. AEG-1 activates NF-κB promoter activity. HeLa cells were infected with Ad.AEG-1 (50 pfu/cell) or treated with TNF-α (10 ng/ml) and 12 hr later were transfected with the indicated plasmids using Lipofectamine 2000. Luciferase assays were performed 48 hr after transfection using a Luciferase Reporter Gene Assay kit according to the manufacturer's protocol. The β-galactosidase activity was determined using the Galacto-Light Plus kit. Luciferase activity was normalized by β-galactosidase activity, and the data presented is the fold-activation relative to pGL3-Basic from triplicate determinations. C. Inhibition of NF-κB blocks AEG-1-induced increased anchorage independent growth. Upper panel, HeLa cells were either uninfected or infected with Ad.AEG-1 or Ad.IκBα–mt32 either alone or in combination at an m.o.i. of 50 pfu/cell for each Ad. Two days later the level of p65 was determined in the nuclear extracts by Western blot analysis. Lower panel, HeLa cells were infected as in the upper panel. Twenty-four hr later, cells (1 × 105) were replated in 0.4% agar on 0.8% base agar. Two weeks later, colonies ≥ 50 cells were counted under a dissection microscope. Data represents mean ± SD of triplicate plates in three independent experiments. D. Inhibition of NF-κB blocks AEG-1-induced enhanced matrigel invasion. Left panel, HeLa cells were infected as in Fig. 4C. Twenty-four hr later, cells (2.5 × 104) were seeded onto the upper chamber of a matrigel invasion chamber system in the absence of serum. Forty-eight hr after seeding the filters were fixed, stained and photographed. Right panel, graphical representation of the invasion assay. Data represents mean ± SD of three independent experiments. Reproduced, by permission of the publisher, from Emdad et al (2006).