Figure 4

Vav1Y3F increases migration of MCF-10A cells by causing secretion of an EGF receptor-dependent factor. (A) MCF-10A cells expressing GFP or Vav1Y3F were lifted and pretreated with hybridoma medium (control) or with mAb225 for 30 minutes at 37°C. Cells were then seeded in transwells in wells containing assay media minus and plus 5 ng/ml EGF for GFP or assay media minus EGF for Vav1Y3F and allowed to migrate overnight. The data are graphed as the percentage of migration of control Vav1Y3F cells in media without EGF (set to 100%) for each individual experiment. Data are the average plus standard deviation for 3 transwells. (B) MCF-10A cells expressing GFP or Vav1Y3F were lifted and pretreated with DMSO (control) or 300 nM AG1478 for 30 minutes at 37°C. Cells were then seeded in transwells in wells containing assay media plus DMSO -/+ EGF or assay media plus AG1478 -/+ EGF as indicated and allowed to migrate overnight. Data are expressed as in panel A and are the average plus standard deviation for 3 transwells. (C) Conditioned media was collected from MCF-10A cells expressing GFP or Vav1Y3F as described in the Materials and Methods. Transwells were placed in wells containing the different conditioned medias and naïve MCF-10A cells were seeded in the top of the transwell. Cells were allowed to migrate overnight and migration was analyzed as in panel A. Data are expressed as the fold migration of cells with migration of cells in GFP cell conditioned media set to 1. Data are the average plus standard deviation of 5 independent experiments.