Tamoxifen citrate

Tamoxifen citrate, as potent synthetic anti-estrogen, was commercially available (Calbiochem, Merck, Darmstadt, Germany; # 579000). According to the manufacturers’ instructions, 50 mg of Tamoxifen citrate was dissolved in 10 mL pure ethanol (J.T. Baker, ThermoFisher Scientific, Waltham, MA, USA). Tamoxifen solution was added to the cell culture medium to treat the cells. The four cell populations (ASC, MCF-10A, MCF-7 and BT-474) were each treated individually, as well as the three co-cultures. Cells were treated with a final concentration of 5 µM, 15 µM and 25 µM tamoxifen for 48 and 96 h and cell culture medium containing tamoxifen was changed daily. Untreated samples served as control.

N-(p-amylcinnamoyl)anthranilic acid (ACA), maintained as a 50 mM stock solution in dimethylsulfoxide (DMSO), 2-aminoethoxydiphenyl borate (2-APB; 75 mM stock solution in DMSO), aristolochic acid (75 mM stock solution in DMSO) and 3-methyladenine (3-MA; 50 mM stock solution in DMSO) were purchased from Sigma (St. Louis, MO, USA). N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG; 0.5 M stock solution in DMSO) was purchased from AccuStandard (New Haven, CT, USA). Doxorubicin hydrochloride and tamoxifen citrate were purchased from Thermo Scientific (Waltham, MA, USA). Q-VD-OPh was purchased from R&D Systems (Minneapolis, MN, USA) and maintained as a 50 mM stock solution in DMSO. Propidium iodide (PI) (10 mg/ml solution) was purchased from Thermo Scientific. ApoScreen Annexin V-fluorescein isothiocyanate (FITC) was purchased from Southern Biotech (Birmingham, AL, USA).