FIGURE 7.

RhoC-induced MDA-MB231 cell migration requires IQGAP1. A, MDA-MB-231 cells were transfected with siRNAs specific for GFP or IQGAP1; cell migration was assessed in a transwell assay with some cells receiving EGF in the lower chamber, as described under “Experimental Procedures.” The number of cells migrating under control conditions (GFP siRNA-transfected, no EGF) was assigned a value of one (*, p < 0.05 for the comparison to control; ns, non-significant). IQGAP knock-down was assessed by Western blotting (below). B, cells were transfected as in panel A, and some cells were stimulated with EGF for 20 min. Endogenous, GPT-bound Rho proteins were isolated by RBD-pulldown assay and absolute amounts of GTP bound to the beads were measured using the luciferase-based assay described under “Experimental Procedures” (**, p < 0.01 for the comparison to control). C, MDA-MB-231 cells were siRNA transfected as in panel A, but were infected with LacZ virus (0) or with two different concentrations of adenovirus encoding HA-tagged RhoC^V14 (1 and 3×). Cell migration was assessed by transwell assay with all cells receiving FBS in the lower chamber. The number of cells migrating under control conditions (GFP siRNA-transfected and LacZ virus-infected) was assigned a value of one (*, p < 0.05 for the comparison to control; #, p < 0.05 for the comparison to GFP siRNA-transfected cells infected with the same amount of RhoC^V14 virus). D, cells were treated as in panel C, and the amount of GTP bound to RhoC^V14 was measured in anti-HA immunoprecipitates as described under “Experimental Procedures” (#, p < 0.05 for the comparison to GFP siRNA-transfected cells infected with the same amount of RhoA^L63 virus). E, cell lysates from cells treated as in panel C were analyzed by Western blotting with antibodies specific for RhoC (top panel, note that the HA epitope-tagged RhoC^V14 migrates with a higher apparent molecular weight than endogenous RhoC), IQGAP1 (middle panel), and β-actin (bottom panel).