Rho- and Rac-dependent morphology of RBE cells during adhesion to laminin-1. (Ai) Serum-starved wild-type (wt) and CEACAM1-L–expressing cells were plated with or without manipulation on laminin-1 for 75 minutes, fixed, and stained for actin filaments with phalloidin-TRITC. During adhesion, cells were treated without (control, -FCS) or with 10% FCS (+FCS) or LPA (50 and 500 ng/mL) or Rho-kinase inhibitor Y27632 (10 μM) to activate or inhibit Rho-dependent signaling pathways. For demonstration of the reversibility of ROCK inhibition, cells were preincubated with Y27632 (10 μM) for one hour and plated in new serum-free medium without the inhibitor (wash-out). Original magnification, × 400. (ii) Protrusion length in RBE/wt (□) and CEACAM1-L–expressing (▪) cells after treatment with FCS, LPA, and Y27632 was measured with AxioVision software. Means ± SD of 2 independent experiments are shown (n ≥ 20). ^*Altered protrusion length of CEACAM1-L–expressing cells by the indicated treatment represents significant difference, P < .001. (Bi) Morphology of RBE cells overexpressing Rac1 wt, L61Rac1 (dominant active), or N17Rac1 (dominant negative) on laminin-1. Wild type (wt) and CEACAM1-L–expressing cells were transiently transfected with myc-tagged Rac1 constructs by nucleofection. Then 8 hours later, serum-starved cells were plated on laminin-1 for 75 minutes, fixed, and stained for actin filaments with phalloidin-TRITC (left panels) and with anti–myc-tag antibodies and anti–mouse Cy2-conjugated secondary antibodies (right panels) for visualization of Rac1-construct expression. Original magnification, × 400. (ii) Protrusion length of untransfected (control) or Rac1-construct–transfected RBE cells was measured with AxioVision software. □ indicates wt; ▪, CEACAM1-L–expressing cells. Results are presented as means ± SD of 2 independent experiments (n ≥ 10). ^*Decrease in protrusion length of CEACAM1-L–expressing cells by dominant-negative Rac1 expression represents significant difference, P < .001.