Figure 8

Src inhibition blocks β₃ integrin and TGF-β mediated MAPK activation, EMT, and invasion in NMuMG cells. (a) Control or β₃ integrin expressing NMuMG cells were serum starved for 4 hours before TGF-β₁ (5 ng/ml) stimulation in the absence or presence of either PP2 (10 μmol/l) or its inactive counterpart PP3 (10 μmol/l). Afterward, the activation status of ERK1/2 and p38 MAPK was determined by immunoblot analysis using phospho-specific antibodies. Re-probing stripped membranes with anti-ERK1/2 antibodies monitored differences in protein loading. Data are from a representative experiment that was repeated twice with similar results. (b) β₃ Integrin expressing NMuMG cells were stimulated with TGF-β₁ (5 ng/ml) for 36 hours in the absence or presence of PP2 (10 μmol/l). Afterward, alterations in actin cytoskeletal architecture were visualized by direct rhodamine-phalloidin immunofluorescence. Shown are representative images from a single experiment that was repeated twice with identical results. (c) Control (GFP) or DN Src-expressing NMuMG cells were stimulated with TGF-β₁ (5 ng/ml) for 24 hours. Afterward, alterations in actin cytoskeletal architecture were visualized by direct rhodamine-phalloidin immunofluorescence. Total Src expression was monitored by probing detergent-solubilized whole cell extracts (50 μg/lane) with anti-Src antibodies, whereas exogenous Src expression was visualized using anti-Myc antibodies. Immunoblotting stripped membranes with anti-β-actin antibodies served to monitor differences in protein loading. Shown are representative images from a single experiment that was repeated twice with identical results. (d) Control (GFP), DN Src, CA Src, β₃ integrin, and β₃ integrin/DN Src 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 number of cells invaded per well. (e) Src-deficient NMuMG cells were generated by siRNA transfection. The effects of Src deficiency on TGF-β mediated EMT were 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 monitor Src expression by immunoblotting with anti-Src antibodies. Differences in protein loading were monitored by re-probing striped blots with anti-β-actin antibodies. CA, constitutively active; DN, dominant negative; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; TGF, transforming growth factor; WB, Western blot; WT, wild type.