Immortalized Mouse Tumor Mammary Fibroblast Protocol

Immortalized mouse tumor mammary fibroblasts were generated in Dr. Harold Moses laboratory (Vanderbilt University), used in previous publications (18 (link)–20 (link)), and gifted to us. Fibroblasts were growing in T-75 flasks (Fisher scientific, USA) in DMEM medium (Gibco, USA) supplemented with 10% FBS and 1% Antibiotic-antimycotic (Gibco, USA). Cell lines were not authenticated. Cells were Mycoplasma-free, tested using LookOut Mycoplasma PCR Detection Kit (Sigma), and used within 20 passages of thawing. For Western blotting assay, fibroblasts were transferred to 10cm cell culture dishes (Corning, USA). When cells achieved 90–95% confluency, they were treated with TGFβ, 1 ng/ml (R&D System, USA) and NECA (5′-N-Ethylcarboxamido adenosine, Sigma-Aldrich, USA).