Rho kinase–mediated myosin II activity and substrate stiffness slow MEF migration and increase adhesion size. (A) Migration rates for untreated cells (control), cells treated with 20 µM blebbistatin (Blebb) or 10 µM Y27632, or plated on 1.0 kPa compliant polyacrylamide substrates. Mean velocity is shown above each box plot. Arrowheads indicate lower and upper extreme outliers. n = number of cells. (B) Phase-contrast images of cell morphology under the same conditions as in A. Bar, 10 µm. (C) Immunolocalization of PY epitopes (P-Tyr) to visualize adhesions (green) and fluorescent phalloidin staining to visualize actin filaments (red) under the treatments as in A. Merged images are shown in the third column, and boxed regions are magnified in the fourth column. Bars: (third column) 10 µm; (fourth column) 2 µm. (D) Area of individual adhesions within PY-immunolabeled cells under the conditions in A. Mean adhesion area (micrometer squared) is shown above each box plot. n = number of adhesions.