Breast Cancer Cell Line Culture and Manipulation

Breast cancer cell lines MCF7 and Bcap37 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Both cell lines were ER positive. Cells were grown in normal DMEM with 10% FBS, 100 unit/mL penicillin and 100 µg/mL streptomycin. Prior to all experiments, cells were pretreated for at least 1 week with an estrogen-free medium (phenol red-free DMEM with 10% dextran-coated charcoal serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 0.5 mM sodium pyruvate, and 2 mM l-glutamine) to prevent the effects of serum-derived estrogenic compounds. Cell transfection was carried out using Lipofectamine 2000 (Cat. 11668, Invitrogen) according to the manufacturer's instructions.
E2 (E8875, Sigma) was prepared as a 10 mM solution with ethanol and stored at −80°C. Tamoxifen (T5648, Sigma) or toremifene (T7204, Sigma) was dissolved in 10 mM DMSO. Phps1 (P0039, Sigma,) was dissolved in PBS solution. NSC87877 (565851, Millipore) and phps4 (a gift from Dr. Yuehai Ke (Department of Medicine, Zhejiang University) were dissolved in DMSO. Plasmids expressing siRNAs of Shp2 Plvth-H1, Plvth-H2, and Plvth-H3, as well as a control plasmid Plvth were also prepared as previously described [53] (link). Unless otherwise indicated, all other chemicals were obtained from Sigma/Fisher.