Figure 5

β₃ Integrin expression blocks TGF-β stimulated growth arrest but enhances TGF-β stimulated invasion and EMT in NMuMG cells. (a) Control (GFP), WT, or D119A β₃ integrin expressing NMuMG cells were stimulated with increasing concentrations of TGF-β₁ for 48 hours. Cellular DNA was radiolabeled with [³H]thymidine and quantified by scintillation counting. Data are the means (± standard error) of three independent experiments presented as the percentage of [³H]thymidine incorporation normalized to untreated cells. (b) Control (GFP) and β₃ integrin expressing NMuMG cells were allowed to invade through Matrigel matrices in the absence or presence of TGF-β₁ (5 ng/ml) for 36 hours. Values are the mean (± standard error) of three independent experiments presented as the percentage invasion relative to TGF-β stimulated β₃ integrin expressing NMuMG cells. (c) The cell morphology of control (GFP), WT, and D119A β₃ integrin expressing NMuMG cells was monitored by phase contrast microscopy, and alterations in actin cytoskeletal architecture was visualized by direct rhodamine-phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. (d) Control (GFP), WT, or D119A β₃ integrin expressing NMuMG cells were transiently transfected with pSBE-luciferase and pCMV-β-gal cDNAs, and subsequently were stimulated with TGF-β₁ (5 ng/ml) for 24 hours. Afterward, luciferase and β-gal activities contained in detergent-solubilized cell extracts were measured. Values are the mean (± standard error) luciferase activities observed in three independent experiments normalized to maximal reporter gene expression measured in unstimulated cells. EMT, epithelial-mesenchymal transitions; GFP, green fluorescent protein; TGF, transforming growth factor; WT, wild type; SBE, Smad binding element.