RhoA activation is essential for Wnt5a-induced MDA-MB-231 cell migration.

(A, B) RhoA activation was induced by Wnt5a (A) and blocked by Dvl2 mutants or siRNA (B). Serum-starved MDA-MB-231 cell monolayers were incubated with 500 ng/mL rWnt5a for 0–60 min (A), or transiently transfected with Dvl2 mutants or siRNA, and then incubated with 500 ng/mL rWnt5a for 30 min (B). Cell lysates were assayed for active RhoA by pulldown assays. Results are presented as mean ± s.d. of 3 independent experiments in (A) and (B). (D) Expression of empty vector, WT-RhoA, V14-RhoA or N19-RhoA was verified using total protein from cells and immunoblotted using anti-GFP antibody. (C, E) Wnt5a-induced cell migration was abolished by C3 exoenzyme transferase (C) or N19-RhoA, a dominant negative mutant of RhoA (E). MDA-MB-231 cells were preincubated with Rho inhibitor C3 (10 ng/µL) for 1 h (C), or transiently transfected with empty vector, WT-RhoA, V14-RhoA, or N19-RhoA (E), and then incubated with 500 ng/mL rWnt5a for 4 h. Cell migration rate was determined by wound healing assay. Results are presented as mean ± s.d. of 5 independent experiments in (C) and (E).