were mounted on microscope slides and imaged with a conventional immunofluorescence microscope or a confocal microscope. Confocal stacks 15210837 were reconstructed with Imaris Software. Molecular Subtyping First, intrinsic subtype classification into Luminal A, Luminal B, Basal-like, HER2-enriched and Normal-like groups was performed using the 50 gene predictor, comparing Py2T and Py2T LT to the UNC337 training set provided by Parker et al.. Briefly, the centroids from 50 intrinsic genes were compared between the training set and the cell lines analysed here using Spearman’s rank correlation to predict the subtype on the test set using PAM50 predictor bioclassifier R script with R2.13.0 . In a second step, Claudin-low subtype prediction was performed as described by Prat et al.. Briefly, centroids for ��claudin-low��or ��others��were calculated on the training set provided by Prat et al. including different breast cancer cell lines from 2559518 the Neve et al. study. For each novel cell line to be classified, the Euclidean distance to the centroids from the training set was calculated and the subtype assigned according to the nearest centroid. Classification was performed using R2.13.0. Immunoblotting Cells were lysed in RIPA buffer containing 2 mM Na3VO4, 10 mM NaF, 1 mM DTT, and a 1:200 dilution of stock protease inhibitor cocktail for mammalian cells. Protein concentration was determined using the BCA assay kit. Equal amounts of protein were diluted in SDS-PAGE loading buffer and resolved by SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes by semi-dry transfer, blocked with 5% skim milk powder in TBS/0.05% Tween 20 and incubated with the indicated antibodies. HRP conjugated secondary antibodies were detected by chemiluminescence using a Fusion F67 chemiluminescence reader. Luciferase Reporter Assay 56104 Py2T cells were plated in triplicate in a 24 well-plate. One day after plating, cells were transfected with 800 ng reporter and 5 ng Renilla encoding plasmids using Lipofectamine 2000. Fresh growth medium was added after 5 hours of transfection containing 2 ng/mL TGFb or not. After 2 days, cells were lysed directly in plates using 16 passive lysis buffer and GSK1363089 site lysates were analyzed using the Dual-Luciferase Reporter Assay System and a Berthold Luminometer LB960. Measured luciferase values were normalized to internal Renilla control. The Smad4 reporter was kindly provided by Dr. P. ten Dijke into the retroviral vector pBabe-hygro. The resulting plasmid pBH-EGFP was transfected into the retroviral packaging cell line Plat-E using FugeneHD. One day after transfection, medium Cell Line Isolation A piece of freshly isolated tumor was transferred into collection medium ) and Py2T EMT Model was exchanged and retroviral supernatant was produced for 2 days. Viral supernatant was filtered through 0.45 mm pores and 8 mg/mL Polybrene was added. Py2T cells were plated into 6well plates and were infected with viral supernatant one day after plating. For infection, 2 mL supernatant was added per well and plates were spun for 1 hour at 30uC at 10006g and were subsequently incubated at 37uC with 5% CO2 in a tissue culture incubator for 2 more hours. Viral supernatant was then replaced by normal growth medium and one day later, selection with 500 mg/mL Hygromycin B 
was performed for 5 consecutive days. 3D Matrigel Culture and In-gel Immunofluorescence Staining Growth factor-reduced Matrigel stock was thawed on ice and diluted to 4 mg/mL protein wit