Figure 7.

Effects of the expression of mutant N-cadherin on β-catenin dissociation and TEM of melanoma cells. (A) WM239 cells were transiently transfected with a myc-tagged wild-type or mutant (Y860F) N-cadherin. The expression of myc-tagged protein was confirmed by immunoblot analysis. The arrow indicates the endogenous N-cadherin, and the arrowhead indicates the expression of myc-tagged wild-type N-cadherin (lane 2) or myc-tagged mutant N-cadherin (lane 3). Control cells are shown in lane 1. (B) The transfectants were cocultured with an endothelial monolayer for 5 h before fixation and double staining using a myc mAb and a rabbit antibody against β-catenin. Myc staining was used to identify transfected cells (asterisks), and the confocal images show only β-catenin staining: melanoma cell expressing myc-tagged mutant N-cadherin (a) and melanoma cell expressing myc-tagged wild-type N-cadherin (b). Arrows indicate the heterotypic contact between transfectants and endothelial cells. Bars, 10 μm. (C) Melanoma cell were transiently transfected with wild-type N-cadherin-myc or mutant N-cadherin-myc and cocultured with an endothelial monolayer in a 3:1 ratio. Samples were collected at 0 or 5 h for immunoprecipitation with an antibody against myc. Protein blots of the immunoprecipitates were probed with antibodies against β-catenin, N-cadherin, and phospho-tyrosine. The pTyr-positive bands correspond to myc-tagged N-cadherin. (D) Transfected cells transiently expressing either wild-type or mutant myc-tagged N-cadherin were subjected to the TEM assay. Transmigration of myc-positive cells was scored at 5 h of coculture. Data represent the mean ± SD (n = 3).