Purified recombinant active tgf β 1

Human MCF10A mammary epithelial cells were obtained from the National Cancer Institute Physical Sciences in Oncology Bioresource Core Facility, in conjunction with American Type Culture Collection (Manassas, VA). Madin-Darby Canine Kidney (MDCKII) cells were a gift of Rob Tombes (VCU). All cells were cultured in a humidified atmosphere at 37 °C with 5% CO₂. MCF10As were maintained under standard culture conditions in DMEM/F-12 HEPES (Life Technologies, Carlsbad, CA), supplemented with 5% horse serum, 0.05% hydrocortisone, 0.01% cholera toxin, 0.1% insulin, 0.02% EGF and 1% antibiotics. MDCKII cells were maintained under standard culture conditions in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% antibiotics. Purified recombinant active TGF-β1 was purchased from Sigma Aldrich (St. Louis, MO). Immunofluorescence imaging was conducted using the following primary antibodies: Ms anti-Hu E-cadherin (HECD-1, Abcam, Cambridge, United Kingdom), Ms anti-Ms N-cadherin (BD Biosciences, San Jose, CA), Rb anti-Hu FN (Abcam, Cambridge, United Kingdom), Ms anti-Hu LTBP-1 (RD Systems, Minneapolis, MN), Rb anti-Hu Smad2 (86F7, Cell Signaling Technology, Danvers, MA), Dapi (Thermo Fisher Scientific, Waltham, MA). F-actin images were acquired by labeling cells with AlexaFluor555 Phalloidin (Life Technologies, Carlsbad, CA).