Fig 1: Establishment of a murine breast cancer cell line undergoing TGFß-induced EMT.(A) Primary tumor cells were isolated from an advanced breast tumor of a MMTV-PyMT transgenic female mouse and were cultured for at least 2 months prior to further experimentation, resulting in a novel cell line termed Py2T. (B) Py2T cells maintain the MMTV-PyMT transgene. The MMTV-PyMT transgene was detected by PCR and agarose gel electrophoresis. DNA from an MMTV-PyMT tumor and from normal murine mammary gland (NMuMG) cells served as positive and negative controls, respectively. (C) Py2T cells lost the expression of the MMTV-PyMT transgene. Immunoblotting for the PyMT protein was performed on lysates of Py2T cells untreated or treated with 0.1 µM Dexamethasone for up to 72 h to induce the MMTV promoter. Lysates of an MMTV-PyMT tumor and NMuMG cells served as positive and negative controls, respectively. (D) Treatment of Py2T cells with known EMT inducers. Cells were continuously treated with the indicated growth factors and cytokines for 10 days (2 ng/mL TGFß1; 50 ng/mL EGF; 10 ng/mL IGF-I; 50 ng/mL HGF; 20 ng/mL FGF-2; 20 ng/mL PDGF-BB; 50 ng/mL IL-6). Potential morphological changes were analyzed by phase-contrast microscopy. (E) Expression of epithelial (E-cadherin) and mesenchymal (N-cadherin, fibronectin) markers were analyzed by immunoblotting of the lysates of cells treated in (D). (F) Immunoblotting analysis of EMT marker expression in Py2T and Py2T LT cells. The mesenchymal subline Py2T LT (long-term) was generated by TGFß-treatment of Py2T cells for at least 20 days, and was subsequently maintained in TGFß containing growth medium. (G) Analysis of markers for EMT and breast cell type before and after TGFß-induced EMT. Immunofluorescence staining was performed with antibodies against E-Cadherin (epithelial marker), vimentin (mesenchymal marker), estrogen receptor alpha (ERa), cytokeratin 8/18 (luminal markers) and cytokeratin 14 (basal marker). Scale bar, 20 µm.