Figure 6.

Inhibition of MEK/ERK, ROCK, and MLCK activities reverses Src-induced deregulation of E-cadherin–associated cell-cell contacts. (A) KM12C/vector or KM12C/Src527F cells were switched from low- to high-calcium medium for 4 h (a and b). MEK inhibitor UO126 (25 μM; c), ERK activation inhibitor peptide II (100 μM; d), ROCK inhibitor Y27632 (10 μm; e), or MLCK inhibitor ML7 (5 μM; f) was added to high-calcium medium and KM12C/Src527F cells were maintained for 4 h in such medium. Cells were fixed and stained with anti–E-cadherin antibody. Solid arrows show accumulation of E-cadherin at cell-cell contacts in KM12C/vector cells and KM12C/Src527F cells treated with either UO126, ERK activation inhibitor peptide II, Y27632, or ML7 (a, c, and d–f). Broken arrow in b points to disrupted E-cadherin staining in KM12C/Src527F cells after the switch to high-calcium medium. Scale bars, 10 μm. (B) Quantitation of percentage of KM12C/Src527F cells that are forming cadherin-mediated cell-cell contacts when cells are switched to high-calcium medium containing ML7, UO126, or Y27632. (C) KM12C/Src527F were plated on fibronectin-coated substratum for 2 or6h(a–f) without inhibitors (a and d) or with the MEK inhibitor UO126 (25 μM; b and e) or MLCK inhibitor ML7 (5 μM; c and f). Cells were fixed and stained with anti–E-cadherin antibody. Arrows show accumulation of E-cadherin at cell-cell contacts in KM12C/Src527F cells plated on fibronectin when MEK/ERK or MLCK activity is inhibited. Scale bars, 10 μm. (D) (a) KM12C/Src527F were plated on poly-l-lysine–coated substratum for 6 h and E-cadherin was visualized by staining with anti–E-cadherin antibody. Arrows indicate accumulation of E-cadherin between active Src527F expressing cells when integrin signaling is suppressed. (b) KM12C/vector cells were plated on fibronectin for 6 h, fixed, and stained with anti–E-cadherin antibody. Arrows show accumulation of E-cadherin at cell-cell contacts. Scale bars, 10 μm.