Rho-kinase inhibitor and dominant-negative RhoA block Wnt-3a-induced migration. (A) H929 cells pretreated with sFRP-1 or Dickkopf-1/2 (Dkk1/2) or (B) phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002 (LY, 10 μM) or Rho-kinase inhibitor Y27632 (10 μM) for 1 hour were plated on polycarbonate pore membranes on which the bone marrow stromal cell line HS-5 was pregrown for 24 hours. Wnt-3a CM containing the corresponding inhibitors was added to the bottom chambers followed by incubation for 4 hours. Cells in the lower chamber were harvested and counted as described in Figure 1. Effect of expression of mutant RhoA-N19 on migration of H929 (C) or OPM-2 (D) cells. Wild-type cells or clones expressing vector (PFB-neo) or mutant RhoA (RhoA-N19 nos. 1, 2, 3, and 5) were subjected to migration assay as described for panel A. Results are shown as mean ± SE (n = 3). Figures are representative of 3 separate experiments, respectively. ^*P < 0.01, ^**P < .001. Cell lysates isolated from the same clones were subjected to 12% SDS-PAGE and blotted with anti-Flag antibody to confirm expression of constructs (bottom).