Z 4 hydroxytamoxifen 4 oht

MCF-7 cells were purchased from ATCC (#HTB-22). The tamoxifen-resistant variant of MCF-7 cells (MCF-7/TamR) was generously provided by Dr. Guandi Wang (Xavier University). The T47D cells were kindly provided by Dr. Ameae Walker (University of California, Riverside). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetalbovine serum (FBS; Invitrogen-Gibco) and penicillin– streptomycin (100 IU/mL) at 37 °C in an atmosphere with 5% CO₂. The MCF-7/TamR cells were continuously cultured in the above-described medium containing 0.10 μM (Z)-4-hydroxytamoxifen (4-OHT, Sigma-Aldrich) for at least 6 months to allow them to develop resistance to the drug with an IC₅₀ of ∼5 μM 4-OHT.^{13 (link)}For other experiments, 4-OHT was dissolved in ethanol at a concentration of 10 mM and stored at −20 °C. For SILAC experiments, [¹³C₆,¹⁵N₂]-l-lysine and [¹³C₆]-l-arginine (Cambridge Isotopes Inc.) or their unlabeled counterparts were added to SILAC DMEM medium depleted of l-lysine and l-arginine (Thermo Scientific Pierce) until their final concentrations reached 0.398 and 0.798 mM, respectively, to yield “heavy” and “light” media. Cells were cultured in the “heavy” SILAC DMEM medium for at least 6 cell doublings to ensure complete incorporation of heavy-isotope-labeled amino acids.

All cell lines used in the study, including mammary epithelial cell lines (human HMLE, MCF10A and mouse NMuMG cell lines), breast cancer cell lines (human MDA-MB-231, HMLE-Neu, T47D, MCF7, BT474, and mouse 4TO7 cell lines) and other cell lines (HEK293T, H29 and HeLa), were cultured using the standard conditions according the American Type Culture Collection (ATCC) instructions. HMLE and HMLE-Neu cells were obtained from Dr. Robert Weinberg at MIT. iMMEC cells were obtained from Dr. Vassiliki Karantza at CINJ. Primary isolated mammary epithelial cells (MECs) were cultured with MEGM (Lonza) and immortalized murine MECs (iMMECs) were cultured as described^{66 (link)}. No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample. The cell lines were not authenticated. Mycoplasma contamination was routinely checked (monthly) in the lab by PCR analysis; all cell lines used in the study were confirmed to be mycoplasma negative. TGF-β1 (R&D systems) and Z-4-hydroxytamoxifen (4-OHT) (Sigma-Aldrich) cell culture treatments in vitro were done for 12 days, at 100 pM and 20 nM, respectively.