Mammosphere Formation Assay Protocol

First, 150,000 cells in 2 ml CGM were plated in a six-well plate. After 12-24 h, cells were treated with EtOH or TAM in the presence or absence of 40 µM CCG-203971 for 6 or 36 h in RGM. Cells were trypsinized with Versene, resuspended in 1 ml 1× DPBS and passed three times through a 25-gauge needle to obtain single-cell suspensions. Then, 10,000 cells were plated in six-well plates previously coated with Poly(2-hydrocyethyl methacrylate) (PolyHEMA) and incubated in 2 ml mammosphere medium composed of 1:1 DMEM/F12, 20 ng/ml human EGF, 40 µg/ml insulin, 500 ng/ml hydrocortisone, 1% penincilin/streptomycin (Merck, 15070-063) and 1:1 Supplement B-27 Minus Vitamin A (Gibco, 12587-010), filtered with a 0.45 µm filter (VWR, 514-4127). Six days after incubation in a 5% CO₂ humidified incubator at 37°C, brightfield pictures were acquired using a 5× objective on a brightfield microscope. Each condition was evaluated in triplicates.