E2 bsa

JKT-1 cells were seeded in six-well plates (0.6 × 10⁶ cells/well). After 48 h, the JKT-1 cells were washed and oestrogen starved overnight in phenol red-free DMEM supplemented with 1% charcoal-stripped FBS. We then added E2 (Sigma-Aldrich^®, Saint Louis, MO, USA), freshly prepared E2-BSA (Sigma-Aldrich^®, Saint Louis, MO, USA) devoid of free E2, which is removed by filtration, ICI-182,780 (fulvestrant; Falsodex^®, AstraZeneca, Wilmington, DE, USA), G1 (Calbiochem^®, Merck KGaA, Darmstadt, Germany), G15 (kindly supplied by Eric R. Prossnitz) [42 (link)], or ethanol (as a vehicle control) at 10⁻⁹ M concentration [15 (link),43 (link)], and incubated them for 24 h. We harvested the cells using trypsin and counted them using the Vi-CELL software (Beckman Coulter, Fullerton, CA, USA). Results are expressed as percentages of variation compared with the control.

E2-BSA (Sigma-Aldrich, V St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS), and free estradiol was removed by filtration using the technique described by Stevis et al. [20 (link)]. The filtered solution was added to serum and antibiotic-free medium. Treatments were carried out in three different concentrations (10⁻¹⁰ M, 10⁻⁹ M, 10⁻⁸ M), and each group consisted of three samples. Estrogen concentrations were calculated with 30 mol steroid per mol bovine serum albumin (BSA) according to manufacturer’s specifications.
17β-Estradiol (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in ethanol then added to serum and antibiotic-free medium in the same three concentrations (10⁻¹⁰ M, 10⁻⁹ M, 10⁻⁸ M). G1 (Tocris Bioscience, Bristol, UK) was dissolved in dimethyl sulfoxide (DMSO) in the final concentration of 10⁻⁸ M. Dynasore (Sigma-Aldrich) was dissolved in DMSO and used in 80 µmol treatment concentration, and was applied 30 min prior to subsequent 17β-Estradiol treatment (in 10⁻¹⁰ M concentration). All treatments were performed in triplicate.
The reagents were added to the cell media for 3 h with the same dissolvent, and then cells were collected and kept at − 80 °C in Trizol^® Reagent (Applied Biosystems, Life Technologies, Carlsbad, California, US) until further processing.