FIGURE 11.

Modulation of spreading and contractility by ROCK and MLCK signaling pathways in NIH 3T3 cells. A, phase-contrast images of NIH 3T3 fibroblast cells cultured on collagen and kassinin substrates for 24 h and subsequently treated with and without 10 μm ML7 (MLCK inhibitor) or 10 μm Y27632 (ROCK inhibitor) for 1 h. Scale bar, 40 μm. B, quantification of cell spreading area in drug-treated cells compared with controls. *, statistical significance (p < 0.05). Error bar, S.E. C, schematic of trypsin de-adhesion assay. Cells were washed with PBS, incubated with warm trypsin, and imaged by time lapse microscopy until cells became rounded (but remained attached to the substrate). De-adhesion time is an indirect measure of cell contractility. D, representative phase-contrast images of cells rounding up upon incubation with warm trypsin. Scale bar, 50 μm. E, de-adhesion times of control and drug-treated cells cultured on collagen and kassinin. Both ML7 and Y-27632 treatment are shown to delay de-adhesion on collagen substrates, whereas on kassinin substrates, cells were sensitive to Y-27632 treatment only. *, statistical significance (p < 0.05). Error bar, S.E.