Deme f12 medium

Breast cancer cell lines MDA-MB-468, MDA-MB-231, and non-tumorigenic epithelial cell lines MCF10A were suspended in serum-free cell freeze and immediately frozen in liquid nitrogen tanks. MDA-MB-468, MDA-MB-231 and MCF10A cell lines were commercially obtained from Beijing Bena Biotechnology Co. (Beijing, China). Cells were cultured in DEME F-12 medium (Gibco, Waltham, MA, USA). Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) was used for transfecting the ACP5 siRNA (Sagon, China) and negative control (NC) into the cells. The target sequences for ACP5 siRNA were TCCTAAATCAAGCATCTTTCTGT (si ACP5).

Porcine GCs from fresh nonatretic follicles (3–5 mm diameter) were isolated and cultured in vitro as described previously.³ Briefly, porcine GCs were extracted by using a syringe with 22‐gauge needle and seeded into 6‐ and 12‐well plates filled with DEME‐F12 medium (#11320033; Invitrogen) containing 10% fetal bovine serum (FBS; #30044333; Gibco), and 1% streptomycin–penicillin, which were then placed in a 37°C incubator with humid atmosphere and 5% CO₂. After incubation for 48 h, cells were firmly attached and prepared for the further in vitro treatment. All the cells used in this study were tested to be uncontaminated and mycoplasma‐negative. For TGF‐β1 and SB431542 treatment, the culture medium were replaced with fresh FBS‐free medium for 10 h, and then TGF‐β1 (#P01137; Novoprotein) and SB431542 (#S1067; Selleck) were added into the culture medium to the final concentration of 10 ng/ml and 10 μM, respectively. For cell transfection, 50 nM small interfering RNAs (siRNAs) and 2.5 μg plasmids were transfected into GCs using Lipofectamine 3000 reagent (#L13778‐150; Invitrogen). The oligonucleotides used in this study were designed and synthesized by GenePharma, which were listed in Table S1.

Vancomycin, ¹³C₆-l-lysine, l-lysine, ¹³C₆¹⁵N₄-l-arginine, l-arginine, Dulbecco’s phosphate buffered saline (PBS), heat inactivate fetal bovine serum (FBS), and dialyzed FBS were purchased from Sigma-Aldrich (St. Louis, MO, USA). DEME/F12 medium was bought from Invitrogen Inc. (Carlsbad, CA, USA). FASP™ protein digestion kit was purchased from Protein Discovery Inc. (Knoxville, TN, USA). The proteomic quantitation kits for acidification, desalting, and digestion, Ionic Detergent Compatibility Reagent (IDCR), DMEM/F12 medium for SILAC, Pierce bicinchoninic acid protein assay kit, and Western blotting substrate were obtained from Thermo Fisher Scientific Inc. (Hudson, NH, USA).