Dextran coated charcoal

A saturation binding assay was conducted [27] (link) using [³H]BPA. The reaction mixture was incubated at 4°C for 2 h with the receptor proteins—GST-fused wild-type ERRγ-LBD or its mutants—in 100 µl of binding buffer [10 mM HEPES (pH 7.5), 50 mM NaCl, 2 mM MgCl₂, 1 mM EDTA, 2 mM CHAPS, and 2 mg/ml γ-globulin]. The assay was performed with or without the addition of unlabelled BPA (final concentration of 1.0×10^-5 M) to quantify the specific and nonspecific binding. After incubation with 100 µl of 1% dextran-coated charcoal (DCC) (Sigma-Aldrich) [28] (link) in PBS (pH 7.4) for 10 min at 4°C, the DCC-absorbed free radio-ligand was removed by the direct vacuum filtration method using a 96-well filtration plate (MultiScreen^HTS HV, 0.45 µm pore size; Millipore, Billerica, MA, USA) for the bound/free separation [27] (link). Radioactivity was determined on a liquid scintillation counter (LS6500; Beckman Coulter, Fullerton, CA, USA).
The data on the specific binding of [³H]BPA were first assessed by means of Scatchard plot analysis [29] (link). Then, these data were applied to a one-site binding hyperbola nonlinear regression analysis by the software package Prism (GraphPad Software Inc., La Jolla, CA, USA) to measure changes in the receptor density B_max and equilibrium dissociation constant K_d. The saturation binding assay was performed at least three times.

MCF-7 breast cancer cells were obtained from the American Type Culture Collection (ATCC) and were grown in high glucose DMEM (4.5 g/L glucose) with stable glutamine, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultured routinely and passaged when reaching a confluence of 80%. For experiments, cells with passage numbers ranging from 5 to 30 were used. For AFM sample preparation, glass slides were rinsed with ethanol and plasma-cleaned for 1 min. Then, 5 × 10⁴ cells were seeded on the glass slides and grown for 24 h prior to treatments. In addition, phenol red-free DMEM supplemented with dextran-coated charcoal (DCC, Sigma) treated FBS (to remove steroid hormones from the serum) was used to test the effect of medium without hormones.
For experiments, cells were either grown in the above specified DMEM (called CTL DMEM) or DCC-treated DMEM without phenol red (called CTL HF). estrogen stocks were prepared by dissolving estrogen (Sigma, St. Louis, MO, USA) in DMSO at a concentration of 1 mg/mL. Cells were treated for 24 h using an estrogen concentration of 100 nM (referred to as DMEM E₂ and HF E₂).

Minimum Essential Medium (MEM), 17β-E₂, E₁, EE and p-nitrophenyl phosphate (pNPP), Dimethyl sulfoxide (DMSO) and dextran-coated charcoal (DCC) were purchased from Sigma, USA. Fetal bovine serum (FBS), fetal calf serum (FCS), antibiotic-antimycotic and L-glutamine were obtained from Gibco, USA. Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) was sourced from Nacalai Tesque, Japan. Glabridin was purchased from Wako, Japan. Phosphate buffered saline (PBS) was from OXOID, USA. Thiazoyl blue tetrazolium bromide (MTT) was obtained from Amresco, USA. Premarin was obtained from Wyeth Pharmaceuticals, USA. Receptor cofactor assay (RCAS) was purchased from Cosmo Bio Co., Ltd. All other chemicals were purchased from Merck, Germany.

VCaP cells were cultured in phenol red-free DMEM (Life Technologies, Darmstadt, Germany) without L-glutamine or pyruvate and supplemented with 10% FBS, 1.25% penicillin/streptomycin, 5% L-glutamine (PAN Technology, Carlstadt, USA), and 2% sodium pyruvate (Life Technologies, Darmstadt, Germany). The medium for VCaP rev cells was supplemented with 1 nM testosterone. The culture medium for VCaP AA cells was supplemented with 5 μM abiraterone acetate hydrolyzed to abiraterone in ethanol/H₂O [22 (link)]. For subsequent experiments, the cells were cultured in medium containing dextran-coated charcoal (Sigma-Aldrich, Taufkirchen, Germany)-treated FBS.

Dexamethasone (Dex), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), dulbecco’s modified eagle’s medium (DMEM), powdered medium, Tris, ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS), sodium orthovanadate, sodium fluoride, protease inhibitor cocktail, dextran coated charcoal, and sodium chloride were all obtained from Sigma (St. Louis, MO). Iron-supplemented bovine calf serum was from Hyclone Laboratories Inc. (Logan, UT). Immobilon-FL polyvinylidenefluoride membrane was obtained from Millipore Corporation (Bedford, MA). Puromycin was from Fisher Scientific (Pittsburgh, PA). FK506 was from Cell Signaling Technology, Inc. (Boston, MA). VX-853 (as timcodar dimesylate) was a gift from Dr. Bruce Gold (Oregon Health and Science University).