Fetal bovine serum (fbs)

MCF-7 cells were maintained in DMEM, 4.5 g/L glucose supplemented with non-essential amino acids (NEAA) (Invitrogen) and 10% fetal bovine serum (FBS) (Biowest). T47D cells were maintained in RPMI 1640 supplemented with NEAA, sodium pyruvate (Invitrogen) and 10% FBS (Biowest). HC-11 cells were maintained in RPMI 1640 supplemented with 2 mM L-glutamine, 5 μg/mL insulin (Invitrogen), 0.01 μg/mL epidermal growth factor (EGF) (Abcys) and 10% FBS (Biowest). MCF10-A cells were maintained in DMEM/F12 supplemented with 0.5 μg/mL hydrocortisone, 10 μg/mL insulin (Invitrogen), 20 ng/mL EGF (Abcys), 100 ng/mL cholera toxin (Sigma) and 5% horse serum (Invitrogen) All cell lines were cultured with penicillin/streptomycin (Invitrogen) at 37 °C under 5% CO₂. For steroid treatments, cells were cultured for at least 24 h in steroids and serum-free DMEM without phenol red and with 2.5% or 5% charcoal/dextran-stripping FBS (Biowest) for MCF-7 and T47D cells, respectively. E2 was purchased from Sigma. Natural enantiomers (6aS and 11aS asymmetric carbon configurations) of glyceollin I and glyceollin II were chemically synthesized by HPC Pharma adapting the synthesis method described by Khupse et al. [17 (link)] and Luniwal et al. [18 ]. The purity was determined at 98% and 99% for glyceollin I and glyceollin II, respectively.

HEK293, MDA-MB-231, Hs578T, MCF-7, Hela, βTC6, HepG2, LLC, HCT-116, and A-431 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Biowest), nonessential amino acids (Gibco), penicillin–streptomycin (HyClone), and GlutaMax (Gibco). A549, H460, H1650, H1299, H358, C4-2B, Du145, LNCaP, 22RV1, Huh-7, MGC-803, MKN-7, and NCI-N87 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS (Biowest), nonessential amino acids (Gibco), penicillin–streptomycin (HyClone), and GlutaMax (Gibco). U2OS and K562 cells were cultured in Iscove’s Modified Dulbecco’s Media (IMDM) supplemented with 10% FBS (Biowest), nonessential amino acids (Gibco), penicillin–streptomycin (HyClone), and GlutaMax (Gibco). The cultured cells were maintained at 5% CO₂ in humidified chamber at 37 ⁰C.

Human embryonic kidney (HEK)‐293T, 293FT, HeLa, and human prostate cancer cell line DU145 were cultured in Dulbecco’s modified Eagle’s medium (Corning, New York, NY, USA) containing 10% FBS (Biowest, Kansas, MO, USA), 1% penicillin, and streptomycin (Invitrogen, Carlsbad, CA, USA). Human breast cancer cell line T47D and SK‐BR‐3 were cultured in RPMI 1640 (HyClone, Logan, UT, USA) containing 10% FBS (Biowest), 1% penicillin, and streptomycin (Invitrogen). Above cell lines were cultured in 5% CO₂ humidified incubator at 37 °C. Human breast cancer cell line MDA‐MB‐231 was cultured in L‐15 LEIBOVITZ (Hyclone) containing 10% FBS (Biowest), 1% penicillin, and streptomycin (Invitrogen) at 37 °C in incubator without CO₂. Cell transfection was performed by using Lipofectamine 2000 (Invitrogen).