Exemestane

Cell lines were routinely maintained in the following types of media containing fetal bovine serum (FBS): DMEM-10% FBS (MCF-7, MDA-MB-231), RPMI-10% FBS (T-47D, ZR-75), McCoys-10% FBS (SK-BR-3) (Gibco, Life Technologies, Grand Island, NY, USA) or Clonetics MEBM + SingleQuot supplements (MCF-10A) (Lonza, Walkersville, MD, USA), plus blasticidin (InvivoGen, San Diego, CA, USA). Experiments, with one exception, were performed in DMEM-10%FBS +/− treatment agents. A subset of experiments was performed in phenol red-free medium containing 5% or 10% charcoal-stripped FBS (Gibco, Life Technologies, Grand Island, NY, USA). Letrozole (Sigma-Aldrich, St. Louis, MO, USA) was prepared as a 50-mM stock solution in 70% ethanol and stored at 4 °C. Anastrozole and exemestane (Selleck Chemicals, Houston, TX, USA) were purchased as 10-mM stock solutions in dimethylsulfoxide (DMSO), and aliquots were stored at −20 °C. Water-soluble 17β-Estradiol (Sigma-Aldrich, St. Louis, MO) was prepared as a 4.29-mM solution in water and stored as aliquots at −20 °C. Testosterone (Sigma-Aldrich, St. Louis, MO, USA) was purchased in compliance with the Stanford University controlled substance program, and prepared as a 1.7-mM stock solution in 70% ethanol stored at 4 °C.

The human BC cell lines MCF-7, SK-BR-3, and MDA-MB-231 were purchased from the China Center for Type Culture Collection (Wuhan, Hubei, China), and all three cell lines were checked for mycoplasma. MCF-7 and MDA-MB-231 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, UT, USA) supplemented with 10% FBS (Lonsera, URY), 100 U/mL penicillin (Procell, Wuhan, Hubei, China), and 100 µg/mL streptomycin (Procell, Wuhan, Hubei, China). SK-BR-3 cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Irinotecan, sunitinib, cisplatin, topotecan, gefitinib, exemestane, and idarubicin (purity >99%) were purchased from Selleck Chemicals (Houston, TX, USA). MTT and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The following primary antibodies were used: anti-SERPINA3 (Cat. ET1705-47, Huabio, China, 1:1000), anti-ANKRD11 (CSB-PA757769LA01HU, Cusabio, China, 1:1000), anti-HDAC3 (ET1610-5, Huabio, China, 1:1000), anti-acetylated H3K9 (AG3948, Beyotime, China, 1:1000), anti-histone H3 (9712, Cell Signaling, USA, 1:1000), anti ERα (8644T, Cell Signaling, USA, 1:1000). RGFP966 was purchased from Macklin (R883017, Macklin, China). Letrozole, exemestane and anastrozole were purchased from Selleck (S1235, S1196, S1188, Selleck Chemicals, China).