Fulvestrant

MCF7 and 293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma−Aldrich) containing 10% fetal bovine serum (FBS) plus 1% penicillin–streptomycin. T47D cells were cultured in RPMI-1640 (Sigma−Aldrich) containing 10% fetal bovine serum with 1% penicillin–streptomycin. Tamoxifen- or fulvestrant-resistant T47D or MCF7 cells were developed by continuous treatment with Tamoxifen (100 nM, > 6 months) or fulvestrant (100 nM, > 4 months), the resistant derivatives were selected when the initially sensitive cells resumed the comparable growth rates to the parental cells, and these cells were cultured in phenol-red free RPMI-1640 medium (Gibco) containing 10% heat-inactivated charcoal-stripped FBS, 1% penicillin–streptomycin and 100 nM 4-OH-Tamoxifen or fulvestrant (48 (link), 49 (link)). Following viral infection, the cells were maintained in the presence of G418 (100 μg/mL) or puromycin (2 μg/mL) depending on the vector. All cells were maintained in an incubator at 37°C and 5% CO₂. 4-OH-Tamoxifen, fulvestrant and etomoxir were obtained from Sigma−Aldrich. DMNQ and TEMPO were purchased from Selleck.

Cells were seeded in 96 well-plates, with or without paclitaxel (#Y0000698, Sigma-Aldrich), doxorubicin hydrochloride (#D2975000, Sigma-Aldrich), tamoxifen (#HT904, Sigma-Aldrich), or fulvestrant (#S1191, Selleckchem). Medium was replaced every two days. After 72 h for paclitaxel and doxorubicin, and seven days for tamoxifen and fulvestrant, to assess the proliferative effect of estrogen, cells were cultured in 24-well plates, in phenol red-free DMEM:F12 medium (# 11039021, Gibco) supplemented with 5% (v/v) charcoal stripped FBS (csFBS, #12676029, Gibco), for five days, with or without 10 nM β-estradiol (#E2758, Sigma-Aldrich). Viability was assessed by Alamar blue assay (#DAL1100, Invitrogen).