Dvl2 activation is required for Wnt5a-induced MDA-MB-231 cell migration.

<p>(A, B) Dvl2 activation was induced by Wnt5a (A) and blocked by sFRP2 (B). Serum-starved MDA-MB-231 cell monolayers were incubated with 500 ng/mL rWnt5a for 0–60 min (A), or treated with 1000 ng/mL sFRP2 for 1 h prior to 500 ng/mL rWnt5a treatment for 5 min (B). Cell lysates were assayed for phosphorylated Dvl2 by immunoblotting analyses with anti-Dvl2 and β-actin antibodies. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 3 independent experiments in (A) and (B). (C) The domain structures of Dvl2 and two mutants. Schematic representations of wild-type Dvl2, ΔPDZ-Dvl2 (lacking the PDZ domain), and ΔDEP-Dvl2 (lacking the DEP domain). Residue numbers above domains denote the domain boundaries. (D) Expression of empty vector, WT-Dvl2, ΔPDZ-Dvl2 or ΔDEP-Dvl2 was verified using total protein from cells and immunoblotted using anti-GFP antibody. (E) Overexpression of ΔPDZ-Dvl2 and ΔDEP-Dvl2 in cells abolished Wnt5a-induced cell migration. MDA-MB-231 cells transiently transfected with EGFP-tagged empty vector, WT-Dvl2, ΔPDZ-Dvl2 or ΔDEP-Dvl2 were incubated in the absence or presence of 500 ng/mL rWnt5a. The cell migration rate was determined by wound healing assays. (F) Efficiency of gene knockdown was analyzed by RT-PCR (left) and immunoblotting (right) for Dvl2. MDA-MB-231 cells were transfected with control (Mock) or Dvl2 siRNAs. Total mRNA or protein extracts from MDA-MB-231 transfected with control (Mock) or Dvl2 siRNA were analyzed by RT-PCR and immunoblotting for Dvl2. The same assay was performed with GAPDH as a loading standard. (G) Dvl2 siRNA significantly inhibited cell migration. MDA-MB-231 cells transfected with control (Mock) or Dvl2 siRNA were subjected to a wound healing assay in the absence or presence of 500 ng/mL rWnt5a. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037823#s3" target="_blank">Results</a> are presented as mean ± s.d. of 5 independent experiments in (E) and (G).