AEG-1 in tumor progression and metastasis. A. AEG-1 expression in various cancer cell lines. Northern blots of the indicated RNA samples were probed with AEG-1 and GAPDH probes. FM516-SV: SV40-immortalized normal human melanocyte; WM35: radial growth phase primary human melanoma; WM238, HO-1, FO-1, SKM p53wt, SKM p53mut, C8161, C8161 6.3, MeWo, 3S5: metastatic and variant human melanoma cells; HMEC: early passage human mammary epithelial cells; HBL-100: immortal normal human breast epithelial cell line; T47D, MCF-7, MDA-MB-157, MDA-MB-231, MDA-MB-453: human breast carcinoma cell lines; NC, normal cerebellum cell line; T98G, G18: human malignant glioma cell line. B. Effect on soft agar colony formation of FM516-SV cells following expression of AEG-1 and Ha-ras, alone or in combination. Upper panel, colony forming ability of FM516-SV cells transiently transfected with the indicated expression vectors. Data shown is average ± S.D. of three independent transfections. Middle panel, colony forming ability of FM516-SV cells stably transfected with AEG-1 and Ha-ras. Data shown is average ± S.D. of three independent transfections. Lower panel, expression of AEG-1 and Ha-ras mRNA in stable transfectants determined by Northern blot analysis. C. AEG-1 is required for Ha-ras-mediated colony formation. THV and THR cells were transfected with control or AEG-1 siRNA using the Lipofectamine 2000 transfection reagent according to the manufacturer's instructions. After 2 days, the cells were trypsinized and counted, and 150 cells were plated in 6-cm dishes. Colonies of ≥ 50 cells were scored after 10 days. The data are represented as mean ± SD and represent three independent experiments in triplicate. D. Ad.AEG-1 infection upregulates NF-κB downstream genes. The expression levels of NF-κB signaling pathway genes after Ad.vec and Ad.AEG-1 infection were analyzed in total RNA using the Human NF-κB signaling gene array (SuperArray Bioscience Corporation) according to the manufacturer's protocol. Graphical representation of expression levels of each gene obtained by densitometric analysis. Reproduced, by permission of the publishers, from Kang et al (2005), Lee et al (2006), and Emdad et al (2006), respectively.