Involvement of Src Family Kinases in N-Cadherin Phosphorylation and -Catenin Dissociation during Transendothelial Migration of Melanoma Cells

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• Figure 1 - Serial confocal images showing melanoma cells at different stages of TEM. Melanoma cells were labeled with Cell Tracker Orange (red) before seeding on an endothelial monolayer cultured on Matrigel. Co-cultures were fixed with paraformaldehyde and F-actin of both cell types was stained with BODIPY-phallacidin (green). Serial confocal images were taken and representative images of melanoma cells at different stages of TEM are shown. Serial images are arranged from top to bottom and the distance of each section to the bottom of the endothelial monolayer is shown in the lower right corner. The side view of the 3D-reconstructed image, together with a schematic drawing, is shown on the top of each panel. (A) A melanoma cell at the initial stage of attachment on top of the endothelium. (B) A melanoma cell with a pseudopodium penetrating the underlying endothelial junction. (C) A melanoma cell transmigrating across the endothelium, with a small part of its cell body still above the endothelium. (D) A transmigrated melanoma cell underneath the re-sealed endothelium. In TEM assay, melanoma cells with spindle-shaped or fibroblastic morphology as illustrated in C and D are scored as transmigrated cells. Bars, 10 μm.

• Figure 2 - Tyrosine phosphorylation of N-cadherin during TEM. (A) Melanoma cells were co-cultured with an endothelial monolayer for 0 h or 5 h. Samples were collected for immunoprecipitation with an antibody against N-cadherin. The immunoprecipates were separated by SDS-PAGE and the immunoblots was probed with antibodies against N-cadherin (arrow), p120ctn (arrowhead), and phospho-tyrosine. (B) Co-cultures were collected at 0 and 5 h for immunoprecipitation with an N-cadherin antibody. The immunoprecipates were eluted from the Protein A-beads with 0.1 ml of 1% SDS. The eluate was diluted 1/10 with IP lysis buffer, and re-precipitated with antibody against either N-cadherin or p120ctn. Blots of these immunoprecipitates were probed using different antibodies. Phospho-tyrosine was detected only in the 5-h N-cadherin band.