Mcf 7

MCF-12A, MCF-10A and MCF-7 were purchased from the ATCC. MCF-12A and MCF-10A cell lines were maintained in DMEM/F12 (Thermo Fisher Scientific) supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF (Invitrogen), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin (Roche) and 1X pen/strep (Wisent). The MCF-7 cell line was maintained in DMEM/F12 with 10% fetal bovine serum (Wisent) and 1X pen/strep.

The human hepatocellular carcinoma cell line Huh7 cells was a kind gift from Dr M. Santo and cultured as previously published (Bagu and Santos, 2011 (link)). Human breast cancer MCF-7 cells were purchased from American Type Culture Collection (American Type Culture Collection). MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (Wisent) supplemented with 10% fetal bovine serum (Wisent) and 1% Penicillin-streptomycin antibiotic. Primary human osteoblasts were isolated and cultured as described (Fendri et al., 2013 (link)). For estrogen assays, cells were cultured in phenol red-free DMEM (Wisent) supplemented with 10% charcoal-stripped fetal bovine serum (CS-FBS) for 3 days. The next day, the cells were treated with ethanol (vehicle) or 10⁻⁷ M E2 for 24 h. E2 and 4-hydroxy-tamoxifen (4-OHT) were purchased from Sigma-Aldrich, ICI-182, 780 was from Tocris Bioscience. E2, along with its inhibitors ICI-182, 780 and 4-OHT, were reconstituted in 100% ethanol as stock solutions of 2×10⁻² M and stored at −20°C as indicated by the manufacturer.

Human BC cell lines MCF-7, MDA-MB-231, and MDA-MB-468 (purchased from ATCC) were used in this study and were routinely verified by STR authentication (Guangdong Hybririo Biotech Ltd, Guangzhou, China). MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Wisent Inc, Montreal, Canada), while the other two cell lines were cultured in L15 medium (GIBCO, Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FBS) (Wisent), at 37°C in a humidified atmosphere with 95% air and 5% CO₂.