Cells from digested mammary glands were seeded at 40,000 cells per well onto MEF’s (for colony formation assays) or 8-well chamberslides (Lab-Tek) coated with 2% Matrigel/DMEM. At the time of seeding, cells were suspended in complete DMEM medium supplemented with vehicle (0.1% BSA/PBS), RANKL (50 ng ml−1), TGFβ1 (5 ng ml−1) or TGFβ2 (5 ng ml−1) (Peprotech). Cells were fixed with 2% paraformaldehyde/PBS after 48 h (chamberslides) or 5 days (colonies) for immunofluorescent staining.
For experiments with MCF10A and HMECs, TGFβ2 stimulations were performed with 5 ng ml−1 TGFβ2 (Peprotech) or 0.1% BSA/PBS (vehicle) for 48 h prior to lysis. In some experiments 30 ng ml−1 of the anti-αvβ3 function blocking antibody LM609 (Millipore) was added to cells at the same time as TGFβ2 or vehicle addition. For the proteasome inhibitor experiments, 10 μM MG132 (Sigma) or DMSO (vehicle) was added to transfected MCF10A cells 5 h prior to lysis. Treatment of MCF10A cells with the Src family kinase inhibitor Dasatinib (Chemietek) or DMSO (vehicle) was performed with 100 nM Dasatinib for the indicated times prior to harvesting lysates.
For experiments with MCF10A and HMECs, TGFβ2 stimulations were performed with 5 ng ml−1 TGFβ2 (Peprotech) or 0.1% BSA/PBS (vehicle) for 48 h prior to lysis. In some experiments 30 ng ml−1 of the anti-αvβ3 function blocking antibody LM609 (Millipore) was added to cells at the same time as TGFβ2 or vehicle addition. For the proteasome inhibitor experiments, 10 μM MG132 (Sigma) or DMSO (vehicle) was added to transfected MCF10A cells 5 h prior to lysis. Treatment of MCF10A cells with the Src family kinase inhibitor Dasatinib (Chemietek) or DMSO (vehicle) was performed with 100 nM Dasatinib for the indicated times prior to harvesting lysates.