Cell culture and establishment of tamoxifen-resistant MCF-7 (TAMR-MCF-7) cells

ER-positive MCF-7 cells were cultured at 37°C in 5% CO₂/95% air in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. TAMR-MCF-7 cells were established using methods previously reported [34, 56] and cultured in DMEM containing 10% charcoal-stripped FBS (Hyclone, Logan, UT) and 4-hydroxytamoxifen (3 μM). For the experiment purpose, the chemicals were exposed in serum free medium condition. For generation of stable knockdown TAMR-MCF-7 cell lines, the lentiviral transduction particles containing short hairpin RNA (shRNA) for Pin 1 (sc-36230-v), or nontarget control shRNA (sc-108080) were purchased from Santa Cruz. Cells were transduced with virus in the presence of polybrene (5 μg/mL) for 24 h and then selected by puromycin (3 μg/mL).