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. 2011 Jul 15;22(14):2423–2435. doi: 10.1091/mbc.E11-04-0306

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© 2011 Wendt et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Down-regulated E-cad expression is required for 3D outgrowth of breast cancer cells. (A) NM-E cells were treated with TGF-β1 (5 ng/ml) for 48 h (Post-EMT) before their 3D culture for an additional 4 d in the absence (NS) or presence of EGF (50 ng/ml) as indicated; 3D outgrowth was monitored by a bioluminescence growth assay. Inset, Immunoblot verifying down-regulation of E-cad in NM-E cells upon TGF-β1induced EMT. Data are the mean (±SE) of three independent experiments completed in triplicate. *, **, p < 0.05. (B) 3D outgrowth of human MDA-MB-231 breast cancer cells (231) expressing E-cad or GFP as a control was monitored longitudinally by bioluminescence. Data are the mean (±SE) of three independent experiments completed in triplicate. Inset, Immunoblot analysis verifying recombinant E-cad expression. (C) 3D outgrowth of murine 4T1 breast cancer cells was monitored by bioluminescence. Data are the mean (±SE) of three independent experiments completed in triplicate. Inset, E-cad protein levels were monitored by immunoblot analyses 5 d after propagating the cells in 2D or 3D cultures. (D) 4T1 cells engineered to stably express an E-cadluciferase reporter construct were grown in the absence or presence of TGF-β1 (5 ng/ml) under 2D or 3D conditions as in (C). Data are the mean (±SE) E-cadfirefly luciferase/CMVrenilla luciferase ratios obtained from three independent experiments completed in triplicate.