Cultivation of Breast Cancer Cell Lines

Human breast cancer cell lines MCF-7, T47D, MCF10A and MDA-MB-231 have been used in our previous studies [31] –[34] (link). MCF-7 and MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (Hyclone, Logan, UT), 100 µg/ml penicillin and 100 µg/ml streptomycin. T47D cells were cultured as previously described [35] (link). MCF10A were cultured in DMEM/F12 (1∶1) containing 5% horse serum (Hyclone, Logan, UT), 100 ng/ml Cholera Toxin, 10 µg/ml bovine insulin, 0.5 µg/ml hydrocortisones (sigma) and 20 ng/ml EGF (Peprotech, Rehovot Israel). Unless otherwise stated, all cell cultures were incubated at 37°C in the presence of 5% CO₂. Cycloheximide (CHX), actinomycin D (Act D) and anti-FLAG antibody were obtained from Sigma. Anti-ERα, anti-HA, and anti-CLOCK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and 17β-estrogen (E2) and ICI 182780 (ICI) were obtained from Abcam (Cambridge, UK).