Z 4 hydroxytamoxifen

MCF-7 and MCF-7-ERα36 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. TAMR-MCF-7 cells were cultured in DMEM supplemented with 10% charcoal stripped FBS (Gemini Bio Product, CA, USA), penicillin/streptomycin and 3 μM (Z)-4-hydroxytamoxifen (Tocris Bioscience, Bristol, United Kingdom). TAMR-MCF-7 cells were established as previously reported (Park et al., 2019 (link)).

Stocks of Rapamycin (R8781, Sigma, St. Louis, MO, USA), Retigabine (SML0325, Sigma), Minoxidil (M4145, Sigma), NS1643 (sc-2041353, Santa Cruz Biotechnology, Dallas, TX, USA), Lamotrigine (L3791, Sigma), Zolmitriptan (SML0248, Sigma), Cariporide (SML1360, Sigma), Topiramate (T0575, Sigma), Pantoprazole Sodium Hydrate (P0021, Sigma), Fenofibrate (F6020, Sigma), Acetazolamide (A6011, Sigma), Quercetin (Q4951, Sigma), Temozolomide (2706, Tocris, Bristol, UK), Dexamethasone (D4902, Sigma), ONO-RS-082 (O0766, Sigma), Topotecan hydrochloride (4562, Tocris), CKD 602 (5125, Tocris), (Z)-4-hydroxytamoxifen (3412, Tocris), Lansoprazole (2582, Tocris), Chlorzoxazone (C4397, Sigma), and Sodium Butyrate (B5887, Sigma), were made at 1000× concentration in DMSO. Stocks of Gabapentin (G154, Sigma) and Cisplatin (232120, EMD Millipore, Burlington, MA, USA) were made in water at 1000×. Dibutyryl cAMP sodium salt (D0627, Sigma) was the only compound dissolved directly in cell culture media at 1 mM concentration.

HoxB8 conditionally immortalized neutrophil progenitors were generated as previously described (20 (link)–22 (link)). Briefly, C57BL/6 mouse bone marrow was isolated, cultured, and transduced with a tamoxifen-inducible Hoxb8 transgene. All cells were grown in Opti-Mem media supplemented with GlutaMax (Gibco), 30uM beta-mercaptoethanol (Sigma-Aldrich), 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), 1x penicillin/streptomycin (Gibco), 1x non-essential amino acids (Gibco) at a density of ~10⁶ cells/mL. The progenitor cell culture was supplemented with stem cell factor (SCF) and 100nM Z-4-hydroxytamoxifen (Tocris Bioscience, Bristol, UK) to induce Hoxb8 expression. SCF was derived from the supernatant of CHO cells that secrete recombinant murine SCF (a gift from Patrice Dubreuil, Centre de Recherche en Cancérologie de Marseille). For differentiation, cells were washed 3x in PBS to remove Z-4-hydroxytamoxifen induction. Cells were pelleted by centrifugation at 400xg for 3 minutes. The differentiation culture was supplemented with SCF and 20 ng/mL G-CSF (BioLegend) for 2-3 days, and then just G-CSF until use on days 5-7.