Breast Cancer Cell Line Maintenance and Hormone Treatments

The breast cancer cell line T47D was obtained from the University of Colorado Cancer Center Tissue Culture core and was maintained in minimal Eagle’s medium, 5% fetal bovine serum (FBS), 1× non-essential amino acids, 1 × 10⁻⁹ M insulin, 0.1 mg/mL sodium pyruvate, and 2 mM L-glutamine. Generation of breast cancer cell lines UCD4 and UCD65 has been previously described [20 (link)]. The UCD4 and UCD65 cell lines were maintained in DMEM/F-12 1:1 with 10% FBS, 1 × 10⁻⁹ M cholera toxin, and 1 × 10⁻⁹ M insulin. Cell lines were authenticated using short tandem repeat (STR) analysis using the University of Arizona Genetics Core (University of Arizona, Tucson, AZ, USA). All cell lines were routinely tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). In vitro hormone experiments were performed using phenol red-free media plus charcoal stripped FBS with the same additives described above. Hormone treatment was used as follows: vehicle (0.2% ethanol), 17-β-estradiol (E2), 10⁻⁸ M (Sigma-Aldrich, St. Louis, MO, USA); R5020, 10⁻⁸ M (PerkinElmer, Waltham, MA, USA); or progesterone (P4), 10⁻⁷ M (Sigma-Aldrich), or the combination of E2 plus R5020 (both 10⁻⁸ M) for 24 h unless otherwise indicated.