TGF-β signaling induces scattering of CA1a cells.
A. Exogenous TGF-β and fibroblast-released TGF-β induces cell scattering in dot assays. Cells were plated into dot assays, incubated over night, and then stimulated with TGF-β (5 ng/ml), EGF (100 ng/ml), or vehicle (Control) for 4 d. Use of CoCM that was derived from cocultures of CA1a cells and TGFβ1 knockout fibroblasts (CoCM(KO)) did not stimulate cell scattering while conditioned medium from cocultures employing TGF-β1 wildtype fibroblasts (CoCM(WT)) did induce cell scattering. B. CoCM as compared to TuCM and FbCM induces higher levels of CAGA (Smad3) and ARE (Smad2) mediated luciferase activity in CA1a cells (n = 3, ANOVA/Dunnett's Multiple Comparison, p = 0.0002 (ARE) and p<0.0001 (CAGA)). C. CoCM contains higher levels of TGF-β than TuCM and FbCM. CCL-64 cells were stimulated with CM overnight and PAI-driven luciferase activity was measured. D. Scattering of CA1a cells is induced by addition of 0.2 ng/ml TGF-β to medium. Cells were plated into dot assays, incubated overnight, and then stimulated with TGF-β (0.2 ng/ml) or vehicle for 4 d.