The construction of cell lines stably expressing GFP-BRK-YF has been previously described (2). Amphotropic HEK293-derived Phoenix packaging cells were used to package pBabe-puro retroviral system. For retrovirus production, packaging cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% bovine calf serum. Transfection with 1% PEI (Polysciences Inc.) was conducted with 10 μg of retroviral DNA in 60 μl of 1% polyethylenimine plus 430 μl of 0.15 M NaCl for the 100-mm culture plates. After 24 and 48 hours, the virus-containing supernatant was collected and filtered through 0.45-μm syringe filter and aliquoted and stored at −80°C. To infect MCF10A, MDA-MB-231, and HEK293 cells, virus-containing supernatant was supplemented with bovine calf serum and polybrene (Sigma-Aldrich, St. Louis, MO) and overlaid on the cells. After overnight incubation, the viral supernatant was replaced with fresh culture medium. Pools of GFP-BRK-YF–expressing cells were selected with puromycin (Sigma-Aldrich). Expression of GFP-tagged BRK-YF was detected after 48 to 72 hours of infection by fluorescence microscopy. To produce stable BRK knockdown cell lines, we used BRK-expressing MCF7 parental cell lines. This knockdown experiment was performed according to the manufacturer’s protocol using shRNA lentiviral vector plasmids from Santa Cruz Biotechnology. The shRNA plasmids generally consisted of a pool of three to five lentiviral vector plasmids, each encoding target-specific 19- to 25-nt shRNAs designed to knockdown gene expression. As controls, MCF7 cells were infected with a control shRNA and a GFP-control plasmid for transfection efficiency. Transfected cells were selected using puromycin (Sigma-Aldrich).

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