Figure 1
TGF-β1 mediated EMT increases cell surface αvβ3 integrin expression in NMuMG cells. NMuMG cells were incubated in the absence or presence of TGF-β1 (5 ng/ml) for 0–36 hours and assayed for EMT. (a) Alterations in actin cytoskeletal architecture were visualized by direct rhodamine-phalloidin immunofluorescence and αvβ3 integrin by indirect immunofluorescence using anti-αvβ3 integrin (LM609) antibodies. Shown are representative images from a single experiment that was repeated twice with identical results. (b) Detergent-solubilized whole cell extracts (50 μg/lane) were prepared to analyze expression of the αv and β3 integrin subunits by immunoblotting. Differences in protein loading were monitored by reprobing striped blots with anti-ERK1/2 antibodies because, unlike the increase in β-actin expression observed (data not shown) ERK1/2 expression was unaltered in NMuMG cells in response to TGF-β1 treatment. (c) β3 Integrin deficient NMuMG cells were generated by siRNA transfection. The effects of β3 integrin deficiency on TGF-β mediated EMT was visualized by direct FITC-phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. Detergent-solubilized whole cell extracts (50 μg/lane) were prepared to analyze expression of the αv, β3, and β1 integrin subunits by immunoblotting. Differences in protein loading were monitored by reprobing striped blots with anti-β-actin antibodies. EMT, epithelial-mesenchymal transitions; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; TGF, transforming growth factor.