Crl 1671

Endometrial adenocarcinoma cell lines, AN3 (ATCC^® HTB-111^™) and RL95-2 (ATCC^® CRL-1671^™) were used for this study. All cells were cultured either in phenol-free DMEM or RPMI medium, supplemented with 10% charcoal stripped FBS (CS-FBS) as well as 1% of penicillin-streptomycin antibiotic (ThermoFisher Scientific, USA). The cells were then cultured in a humidified atmosphere with 5% CO₂ at 37°C until confluent. Cells were then plated in twelve-well culture plates and upon 80% confluence, they were primed with 0.01μM of β-estradiol (E treatment) for 24h. Followed by the addition of progesterone (1μM) to the estrogen primed (EP treatment) wells for 24, 48 and 72h. Ethanol at a concentration of less than 0.01% was used as control (C treatment). The treatment solutions were prepared using commercially available powdered concentrates (Sigma- Aldrich, USA) and dissolved in analytical grade ethanol. The final concentrations were prepared in culture media and stored at -80°C until further use.

Cancer cell lines, including RL95-2 cells (ATCC^® CRL-1671), Ishikawa cells (ECACC, 99040201), MCF7 cells (ATCC^® HTB-22), BT-474 cells (ATCC^® HTB-20), A549 cells (ATCC^® CCL-185) and MGC-803 cells (BLUEFBIO, BFN608007257) were obtained from American Type Culture Collection (Manassas, VA, USA) and European Collection of Authenticated Cell Cultures (Porton Down, UK). These cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) which contained 1% penicillin–streptomycin solution (Gibco, USA) and 10% FBS. The cultured condition was at 37˚C in 5% CO2 humidified atmosphere (HERAcell 150i/240i, Thermo, USA). STR profiling and mycoplasma contamination were performed to keep the authenticity of cell line on regular basis.