4 hydroxy tam

HR+ human BC cell lines (MCF7 and BT474) were obtained from the American Type Culture Collection (Manassas, VA, USA). MCF7 and BT474 cells were routinely grown in RPMI-1640 and DMEM medium, respectively, supplemented with 10% fetal bovine serum, 2 mM glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and 100 unit/ml penicillin/streptomycin. Additionally, 0.1 unit/ml insulin was added to the culture medium for BT474. 4-hydroxy TAM was purchased from Sigma-Aldrich, USA. The TAM-resistant cell lines, MCF7-TAMR and BT474-TAMR, were generated by continuous exposure of MCF7 and BT474 cells to increasing doses of TAM up to 13 μM in 6 months and thereafter maintaining them in presence of 13 μM TAM. TAM resistant cells were cultured in the absence of TAM for 3 days to avoid the influence of TAM in subsequent experiments. Rabbit polyclonal antibody against FRS2 and mouse monoclonal antibody against GAPDH were obtained from Santa Cruz biotechnology, USA.

All mouse housing, breeding, and surgical procedures were approved by the governmental institutions of Baden-Württemberg (Regierungspräsidium Tübingen). BM cells from WT (n = 7, female), Il7rα^Δ (n = 7, female), Il7rα^fl/fl (n = 7, female), Cxcr4^fl/fl (n = 3, female) and FoxO1^fl/fl (n = 3, female) mice were collected and retrovirally transformed with either an empty pMIG vector or with a pMIG vector expressing BCR-ABL1. Unless mentioned otherwise, cells were cultured for 3–7 days in Iscove’s medium (Biochrom AG) containing 10% heat-inactivated FCS (Sigma-Aldrich), 2 mM l-glutamine, 100 U/ml penicillin (Gibco), 100 U/ml streptomycin (Gibco), and 50 µM 2-mercaptoethanol. The medium was supplemented in excess with the supernatant of J558L plasmacytoma cells stably transfected with a vector encoding murine IL7. Transformed cells were selected by IL7 withdrawal and kept in optimum conditions^{38 (link)}. Retroviral vectors containing either constitutively active STAT5 (STAT5-CA)^{34 (link)} or an empty vector (EV) were used to transduce BCR-ABL1-transformed cells and sorted cells were used then for western blot or flow cytometry analysis. 1–2 µM 4-hydroxy Tam (Sigma-Aldrich) was used to induce deletion on plasmids expressing Tam-inducible form of Cre (Cre-ER^T2)^{14 (link)}. All cells were tested and found free from mycoplasma.

4-hydroxy-TAM (#H7904, Lot#086M4013V) and β-estradiol (E₂; #E2257) were obtained from Sigma-Aldrich (St. Louis, MO, USA). D4476 (#74014) was purchased from Stem Cell Technologies Inc. (Cambridge, MA, USA). Antibodies against progesterone receptor (PR; #8757), HER2 (#2242), phospho-ERα (p-ERα) (Ser¹¹⁸, #2511, Ser¹⁶⁷, #64508;), AKT (#4691), phosphor-AKT (Tyr³⁰⁸, #4060), mTOR (#2972), phosphor-mTOR (Ser2448, #5536), PI3 kinase p85α (#13666), phosphor-PI3 kinase p85 (Tyr⁴⁵⁸)/p55 (Tyr¹⁹⁹) (#4228), S6 ribosomal protein (rpS6) (#2217), phosphor-rpS6 (Ser^235/236) (#4858), ERK1/2 (#4695), and phosphor-ERK1/2 (#4370) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against CSNK1G2 (#GTX33123) and ERα (#PA1-309) were obtained from GeneTex Inc. (Irvine, CA, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Tubulin α antibody (#BS1699) was purchased from BioWorld Technology Inc. (Louis Park, MN, USA).