lterations or junction dissolution in cells depleted of Smad4 after one day of TGFb treatment, indicating that canonical TGFb signaling is not required for the initial changes in cell morphology. Non-canonical signaling by TGFb involves the activation of p38 and Jnk MAP kinases via activation of Tak1 by receptor-associated TRAF6 and of Erk1/2 MAP kinase by recruitment and phosphorylation of Shc by TGFbRI and subsequent activation of MEK1/2. These mediators have been well established to contribute to TGFb-induced EMT. Indeed, inhibition of these pathways by chemical inhibitors was sufficient to at least partially block the pronounced morphological changes observed after one day of TGFb treatment. In addition, other non-canonical TGFb-induced signals are known to contribute to EMT, such as RhoA degradation at cell junctions, which results in junction disassembly. We indeed observed a slight decrease in total RhoA expression levels after one day of TGFb treatment. We experimentally mimicked TGFb-induced downregulation of RhoA by siRNA-mediated knockdown in epithelial Py2T cells, which resulted in a partial disruption of tight and adherens junction. Together, these results illustrate that short-term TGFb treatment of Py2T cells evokes cell-cell junction disassembly and pronounced phenotypic changes mainly by non-canonical TGFb signaling. Migratory and Invasive Properties upon EMT Induction To evaluate whether Py2T cells could be a suitable in vitro model system to study functional consequences of EMT, we assessed the migratory and invasive capabilities of these cells before, during and after EMT. First, we employed a modified Boyden chamber assay to analyze whether and to what extent Py2T cells become migratory and invasive during EMT. 
Cells 15647369 previously treated with TGFb for different times were seeded into Boyden chamber inserts without or with Matrigel coating and were allowed to move towards a gradient of fetal bovine serum. Quantification of cells that traversed the membrane revealed that cells treated with TGFb for seven or more days were more migratory compared to untreated cells, and the migratory capacity dramatically increased with longer TGFb treatment. Similarly, when seeded into Boyden chambers pre-coated with Matrigel, the cells passed through the bottom of the chambers with a similar increase over the time of TGFb treatment. To illustrate these results, we stained cells located on the bottom side of 15313368 the insert membranes with crystal violet. These findings clearly demonstrate that Py2T cells display a dramatic increase in chemotactic, single cell migration and invasion upon induction of EMT. Scratch wound buy 64048-12-0 closure is another frequently used assay to assess the migratory capacity of cells on tissue culture plastic. Untreated and TGFb-treated Py2T cells were grown to confluence and then starved in serum-free medium. After scratching a gap into confluent monolayers, we followed gap closure by live cell imaging. Invasive Tumor Formation upon Orthotopic Transplantation into Syngeneic Mice We next orthotopically transplanted Py2T cells into mammary fat pads of mice to evaluate their tumorigenicity. Since Py2T cells have been derived from tumors of MMTV-PyMT mice in an FVB/N background and because the PyMT transgene was no more expressed in cultured cells, we transplanted Py2T cells into syngeneic FVB/N mice. Three mice were injected with 16106 cells, all of which developed tumors. After 27 days of Py2T EMT Model growth, tumors were har
