Cs fbs

Human BC MCF-7 cells were obtained from the Cell Resource Center for Biomedical Research (Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan) and cultured in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries) containing 10% fetal bovine serum (Sigma-Aldrich) and Antibiotic–Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere with 5% CO₂ at 37 °C. HER2-overexpressing MCF-7 clone 5 (HER2-5) cells stably expressing HER2 have been previously established [18 (link)]. HER2-5 AHR-knockout cells (HER2-5/AHRKO) were generated as previously described [17 (link)]. For cell stimulation, HER2-5 cells were cultured in low serum medium containing 2% csFBS (Sigma-Aldrich) for 24 h and treated with recombinant HRG (50 ng/mL).
For the knockdown experiment, HER2-5 cells were transfected with AHR siRNA (Stealth™ RNAi, AHR-HSS 100338; Thermo Fisher Scientific) or control siRNA (Stealth™ RNAi siRNA Negative Control, Thermo Fisher Scientific) using Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. After 24 h, the culture medium was changed to low-serum medium, and the cells were cultured for 21 h. Cells were stimulated with HRG (50 ng/mL) or vehicle (water) for 3 h.

Immortalized human endometrial stromal cells (hESC) were purchased from the American Type Culture Collection (ATCC CRL-4003TM) and cultured according to the manufacturer’s instructions^{73 (link)}. Briefly, stromal cells were cultured in DMEM/F12 (Sigma) supplemented with 10% charcoal-stripped FBS (CS-FBS, Biological Industries) at 37 °C in a humidified chamber with 5% CO2. To induce decidualization in vitro, stromal cells were treated with 1 μM Medroxyprogesterone 17-acetate (MPA, Sigma) and 0.5 mM dibutyryl cAMP (db-cAMP, Sigma) in DMEM/F12 with 2% CS-FBS for 6 days. The medium was changed every 48 h. Under in vitro decidualization, stromal cells were treated with 0.032, 0.16, 0.8, 4, and 20 μM P (Sigma) for further analysis, respectively. The highest treatment dose of P has no significant toxic effect on cell viability.

Immortalized HESC line was purchased from the American Type Culture Collection (ATCC CRL-4003) and cultured according to the manufacturer’s instructions (Krikun et al., 2004 (link)). The identity of the cell lines has been authenticated by ATCC, and we have confirmed that the cell lines tested negative for mycoplasma contamination. Briefly, HESC was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco) supplemented with 1% penicillin and 1% streptomycin, 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries), 3.1 g/l glucose, 1 mM sodium pyruvate, 1% Insulin-Transferrin-Selenium (ITS) (Gibco), and 500 ng/ml puromycin at 37°C in a humidified chamber with 5% CO₂. To induce decidualization in vitro, HESC was treated with differential medium (DMEM/F12 with 2% CS-FBS) containing 1 μM medroxyprogesterone 17-acetate (MPA, Sigma, M1629), and 0.5 mM dibutyryl cAMP (dbcAMP, Sigma, D0627). The medium was changed every 48 hr. Other reagents including inhibitors, activators are listed in Supplementary file 1B.

MCF-7 HTB-22^® cells were obtained from the American Type Culture Collection. Prior to E2 deprivation/re-stimulation experiments, MCF-7 were conditioned for two weeks to phenol-red-free DMEM/F-12 supplemented with 10% (v/v) charcoal-stripped fetal bovine serum (csFBS, Sigma-Aldrich), 1% nonessential amino acids, sodium pyruvate and penicillin/streptomycin (Life Technologies) and to a daily addition of 10 nM E2 in 0.1% DMSO (Sigma-Aldrich) at 37°C, in a humidified and 5% CO₂-enriched atmosphere. For all assays, control cells (CTR) were cultured continuously for 14 days in the above-mentioned conditions, while E2-deprived cells (E2D) were cultured in the same conditions only lacking E2. The re-stimulated cells (ReSt) were E2-deprived for 4 days and re-stimulated with E2 for the 10 following days. Two hours before being harvested, CTR and ReSt cultures were incubated with 10 nM E2 in 0.1% DMSO while E2D were incubated only in 0.1% DMSO. MCF-7 cells that were used as standard controls (STD) as well as in the inhibition and siRNA assays were maintained in the same composition of medium as mentioned above, but with 10% of standard FBS (Eurobio).

Immortalized HESCs cell line, a telomerase immortalized benign endometrial stromal cell line, was obtained from the American Type Culture Collection (ATCC, USA) (Krikun et al. 2004 (link)). HESCs were then cultured as described previously (Liao et al. 2015 (link)). Specifically, cells were cultured at 37 °C with 5% CO₂ in a humidified chamber in DMEM/F12 (Sigma, USA) containing 1 mM sodium pyruvate and 3.1 g/l glucose, supplemented with 10% charcoal-stripped fetal bovine serum (CS-FBS, Biological Industries, Israel), 1% insulin-transferrin-selenium (ITS, Gibco, UK), 500 ng/ml puromycin (Sigma, USA), and 1% penicillin streptomycin (PS, Gibco, UK). As for induction of decidualization, HESCs were cultured in 2% DMEM/F12 media containing 2% CS-FBS as well as 1% PS overnight before treatment with differentiation medium supplemented with 1 mM medroxyprogesterone acetate (MPA, Sigma, USA) as well as 0.5 mM dibutyryl cAMP (db-cAMP, MCE, USA) according to previous report (Brighton et al. 2017 ). The medium was replaced every 2 days.

Immortalized HESC line was purchased from the American Type Culture Collection (ATCC^R CRL-4003^TM) and cultured according to the manufacturer’s instructions22 (link). Briefly, HESCs were cultured in DMEM/F12 (Sigma) supplemented with 10% charcoal-stripped FBS (CS-FBS, Biological Industries) at 37 °C in a humidified chamber with 5% CO₂. Synchronization of HESCs in the G0/G1 phase was achieved by serum starvation overnight as described previously23 (link). To induce decidualization in vitro, HESCs were treated with differential medium (DMEM/F12 with 2% CS-FBS) containing 10 nM E2 (Sigma), 1 μM Medroxyprogesterone 17-acetate (MPA, Sigma), and 0.5 mM dibutyryl cAMP (dbcAMP, Sigma). The medium was changed every 48 h. Thiostrepton (Enzo) and pyridone 6 (Merck Millipore) were used to inhibit FoxM1 and the Janus kinase-STAT3 pathway in culture, respectively.

All cell culture media and supplements except charcoal-stripped foetal bovine serum, CsFBS (Sigma-Aldrich) were obtained from Life Technologies (Paisley, U.K.). Hep3B (ECACC 86062703) and HEK293 (ECACC 85120602) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) through Sigma-Aldrich. MCF-7 cells were kindly provided by Matilda Katan and Ivan Gout (University College London, U.K.). All cell lines (passage numbers <20) were routinely cultured in DMEM (with 25 mM HEPES, GlutaMAX-1 and 4.5 g/l glucose), supplemented with 10% FBS and antibiotics and antimycotics. For transfections, cells were passaged in phenol red-free DMEM supplemented with 10% CsFBS without antibiotics and antimycotics, and seeded at a confluence of 80–90% in 24-multiwell uncoated plates (Hep3B and MCF-7) or on BioCoat poly D-lysine plates (HEK293); both sets of plates were obtained from Corning (Appleton Woods, U.K.).