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. 2012 Jul 12;33(10):1965–1975. doi: 10.1093/carcin/bgs227

Fig. 2.

Fig. 2.

EGF signaling modulates TGF-β signaling to upregulate genes associated with an invasive phenotype in primary prostate epithelial cells. (A) Expression of Vimentin, Slug, Twist2, MT-MMP-1, MMP-2 and MMP-9 genes in IBC-10a (left), PCa-20a (middle) and PC3-ML cells (right). Effects of EGF (10ng/ml) (light gray), TGF-β (10ng/ml) (dark gray), and EGF + TGF-β (10ng/ml each) (E + T) (black) treatments on gene expression was determined by qRT–PCR. Values were normalized to cyclophilin-A and represent the fold increase in expression relative to cells treated with Km (white). Data represent the mean ± SD from three independent experiments. Statistical significance comparing TGF-β treatment alone with E + T was determined using Welch’s unpaired t-test, *P < 0.05, **P < 0.01; ***P < 0.001; #, no significant difference (P > 0.1). (B) Western blot analysis of Slug and Twist2 in IBC-10a cells treated 9 days with Km, EGF, TGF-β or E + T. Tubulin served as the loading control. (C) Representative gelatin zymographs of conditioned media from IBC-10a, PCa-20a and PC3-ML cells treated with EGF, TGF-β or E + T. Equal amounts of protein from conditioned medium from each treatment was resolved by 10% PAGE containing gelatin-A (2mg/ml). Coomassie Blue staining reveals relative levels of enzyamtically active MMP-9 homodimer (210kDa), MMP-9 (92kDa) and MMP-2 (72kDa) in media. (D) Modified Boyden chamber invasion assays showing cell migration following treatment with Km, EGF, TGF-β or E + T for 48h. Representative image of cells on bottom of chamber stained with brilliant blue dye. (E) Prostasphere culture showing three-dimensional acinar structures formed by IBC-10a cells grown as single-cell suspensions in Matrigel for 12 days in the presence of different growth factors Top panel: phase contrast images show cells grew as rounded spheres in the presence of Km, EGF and TGF-β. In contrast, cells grown in the presence of E + T were irregularly shaped and cells invaded the surrounding Matrigel (see red arrows). Lower panel: immunofluorescent labeling of prostaspheres with Vimentin antibodies (green); nuclei labeled with propidium iodide (×200 magnification).