Effects of inhibiting Rac1 or myosin II on stress fiber and focal adhesion densities in cells migrating on 2D surfaces. CHO-α4WT, CHO-α4Y991A, or CHO-α4S988A cells were plated on VCAM-1–coated coverslips in the presence of vehicle control (A, a–c; and D, a–c), NSC23766 (A, d–f; and D, d), or blebbistatin (A, g–i; and D, e). (A) Cells were stained with TRITC-conjugated phalloidin and imaged by confocal microscopy. (B and C) Stress fiber density was measured and graphed as the percentage of total cell area occupied by stress fibers in wild experiments. *, P < 0.05 relative to control. (D) Cells were stained with an antibody against pY-paxillin and imaged by TIRF microscopy. (E and F) Focal adhesion density was measured as the number of focal adhesions (FA) per micrometer squared in the absence (E) or presence of NSC23766 for CHO-α4Y991A or blebbistatin for CHO-α4S988A cells (F). Data represent means ± SEM of 45 cells from three independent experiments. *, P < 0.05 relative to control. Ctrl, control.