Dcc fbs

MCF-7 (purchased from American Type Culture Collection (ATTC, Manassas, VA, USA)), LCC2, LCC9 and LY2 (provided by Robert Clarke, Georgetown University) [15 (link), 28 (link), 29 (link)] cells were grown in phenol red IMEM supplemented with 5% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). These cells represent a model of progression to endocrine/TAM- resistance [30 (link)]. For the experiments described here, cells were grown in hormone depleted medium: phenol red-free IMEM supplemented with 5% dextran-coated charcoal-stripped fetal bovine serum(DCC-FBS, Atlanta Biologicals, Lawrenceville, GA, USA) for 48 h prior to experiments to reduce basal hormone-related activities [31 (link)]. Where indicated, cells were pretreated with 10 μg/ml actinomycin D (ACTD, a transcriptional inhibitor, Sigma, St. Louis, MO, USA) or 100 nM fulvestrant (ICI 182,780; Tocris, Ellisville, MO, USA) for 6 h prior to treatment. Treatments included vehicle control (ethanol (EtOH) or DMSO) or 100 nM 4-hydroxytamoxifen, (4-OHT; Sigma St Louis, MO, USA).

HEK293 cells were purchased from the American Type Culture Collection (ATCC, VA, USA) and cultured at 37°C under a 5% CO₂ atmosphere in a humidified incubator. A phenol red-free version of Dulbecco’s Modified Eagle’s Medium (DMEM/High Modified) (Hyclone, GE Lifesciences, MA, USA) was used as the culture medium. One percent penicillin/streptomycin (Corning, NC USA) and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; Atlanta Biologicals, MN, USA) were supplemented for all cultures. Fifty micrograms Zeocin/ml (Thermo Fisher Scientific, MA, USA) and 400 μg G418/ml (Calbiochem, MA USA) were supplemented during clonal selection of cell lines. Twenty-five micrograms Zeocin/ml and 100 μg G418/ml were supplemented for routine maintenance after selection. No antibiotics were supplemented during compound testing. Human adrenocortical carcinoma H295R cells were purchased from ATCC and cultured at 37°C and 5% CO₂ in a humidified incubator. DMEM-F12 medium (Gibco, MA, USA) was used as the culture medium. Nu-Serum I 2.5% (Corning) and 1× ITS+Premix (Corning) were supplemented for all cultures. Before use in steroidogenesis assays and or freezing, cells were maintained for five passages.