MCF-10A cells expressing ER-Src (MCF-10A–ER-Src cells) were grown in DMEM/F-12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12) with 5% charcoal-stripped fetal bovine serum (Sigma-Aldrich) and supplements as previously described (19). To induce transformation, cells were treated with 1 μM 4-hydroxy-tamoxifen (Sigma-Aldrich) or ethanol as vehicle control for 24 hours. Human embryonic kidney (HEK) 293T and MCF7 cells were grown in DMEM with 10% fetal bovine serum (Sigma-Aldrich). For the soft agar assay, cells (2.5 × 104) were washed with phosphate-buffered saline (PBS) and treated with trypsin. The trypsinized single cells were resuspended in the upper agar phase (growth medium containing final 0.4% agar, freshly prepared at 37°C), which was layered onto the lower agar phase (growth medium containing 1.0% agar). The solidified agar was covered with growth medium, which was changed every 3 days. Colonies (>0.01 mm2) were counted with ImageJ after 30 days.

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