Expression of Wnt-1, -3a, and -4 enhances IGF-I-mediated migration of MM cells. (A) H929 and OPM-2 cells were transfected with empty vector or plasmid constructs containing Wnt-1, -3a, and -4 cDNAs with an HA-tag as described in “Material and methods.” Cell lysates were resolved on 12% SDS-polyacrylamide gels, proteins transferred to membranes and blotted with anti-HA antibody to confirm Wnt expression (top). Cytosolic fractions from cell lysates of positive clones were resolved on 8% SDS-polyacrylamide gels, transferred to membranes, and blotted with anti-β-catenin antibody (middle). The same fractions were also blotted with antitubulin as a control for protein loading (bottom). (B) Positive clones expressing Wnts (H929/Wnt-3a and OPM-2/Wnt-1, -4) or empty vector were starved in serum-free medium for 12 hours. Cell lysates were prepared and incubated with GST-Rho binding domain glutathione beads. Beads were collected by centrifugation, and the bound proteins were analyzed by Western blotting using RhoA-specific antibodies. The same lysates were subjected to 12% SDS-PAGE and blotted with anti-RhoA as a control for protein loading. (C) H929 cells expressing Wnt-3a (H/Wnt3a) or empty vector (H/EV) and OPM-2 cells expressing Wnt-1 (O/Wnt1 no. 3, no. 9) or Wnt-4 (O/Wnt4 no. 1, no. 4) or empty vector (O/EV) were starved in serum-free medium for 3 hours and added to chambers containing polycarbonate pore membranes (5-μM pore size) on which HS-27A stromal cells were pregrown for 24 hours. Growth medium containing IGF-I (12.5 ng/mL) was added to the lower chamber, and after 4 hours incubation cells in lower chamber were harvested and counted. The results are shown as mean ± SE (n = 3). Results are representative of 3 independent experiments. ^*P < .05, ^**P < .01 versus cells transfected with empty vectors.