Figure 2

Cell adhesion induces ligand-independent activation of Met and EGFR. (a) A431 cells were suspended in serum-free medium with (+) or without (-) 2.5 mM EGTA with constant rotation for 24 hours. Representative images from suspended cells are shown. Scale bar, 150 μm. An equal amount of whole cell lysates was analysed by immunoblotting. (b) A431 cells were seeded on culture dishes for 24 h, referred as attached cells (att). The attached cells were trypsinized, suspended in serum-free medium for 30 min, and lyzed (sus). For replating experiments, the suspended cells were replated on dishes coated with 10 μg/ml collagen for various times. The percentage of cell spreading (S) in total counted cells (n ≥ 200) was measured. Representative images are shown. Scale bar, 50 μm. An equal amount of whole cell lysates was analysed by mmunoblotting with antibodies as indicated. (c) A431 cells were suspended and then replated on dishes coated with 100 μg/ml poly-L-lysine (PLL), 10 μg/ml collagen (Col), or fibronectin (FN) for 60 min. The percentage of cell spreading (S) in total counted cells (n ≥ 200) was measured. Representative images are shown. Scale bar, 50 μm. (d) The attached A431 cells stably expressing shRNAs specific to integrin β1 (sh-Integrin β1) or luciferase (sh-Luc) were suspended and replated on dishes coated with collagen for 60 min. An equal amount of whole cell lysates from attached (att), suspended (sus), and replated (rep) cells was analysed by immunoblotting with antibodies as indicated. Scale bar, 50 μm. (e) A431 cells were suspended and then replated onto a culture dish (on dish) or a layer of polyacrylamide gel (on PAA) with elastic moduli of 2.8 kPa, which mimics the stiffness of soft tissues, in the medium with 1% serum for 3 h. Representative images are shown. Scale bar, 50 μm.