Mcf10a cell lines

The human MCF-7 breast cancer and non-tumourigenic MCF10A cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 cells were grown in phenol-red-free RPMI 1640 with L-glutamine (Nacalai Tesque, Kyoto, Japan), supplemented with 10% foetal bovine serum (FBS) (PAA, Pasching, Austria) and 1% penicillin–streptomycin (PAA, Pasching, Austria). MCF-10A cells were cultured in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (PAA, Pasching, Austria), 20 ng/mL epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL cholera toxin (Sigma-Aldrich, St. Louis, MO, USA), and 10 μg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA). The cells used for each experiment were of less than 20 passage number.

MDA-MB-231 cell lines and MCF 10A cell lines were collected from American Type Culture Collection (ATCC) and maintained according to the vendor’s recommendations. Briefly stated, MDA-MB-231 was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, USA), which includes 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 1% penicillin–streptomycin, and high glucose. The special medium obtained from Procell (Wuhan, China; CM-0525) was used to culture MCF10A. In addition, the incubation conditions were set to 37 °C and 5% CO2 for all the cells. The RNAs were isolated from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was conducted in accordance with the manufacturer’s instructions (Takara, Jiangsu, China). Subsequently, the SYBR Green method (Vazyme, Jiangsu, China) was used to assess the target genes’ expression in triplicate. The data analysis was performed by the QuantStudioTM 5 Real-Time PCR System (Thermo Fisher, MA, USA). The cycle threshold (CT) (2^−ΔΔCT) method was employed to calculate the data. The expression levels were normalized to that of β-actin with the comparative CT method. The Table S2 lists the primers used in this study. Besides, the Human Protein Atlas database (HPA, https://www.proteinatlas.org) was employed to validate the protein expression of the genes through immunohistochemistry data.

MCF10A cell lines were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12) supplemented with 5% Horse Serum (Invitrogen), 10 μg/ml insulin, 100 ng/ml Cholera toxin (Sigma Aldrich), 20 ng/ml Epidermal Growth Factor, 1% Penicillin-Streptomycin (Cellgro), and 0.5 μg/ml Hydrocortisone. All cells were grown at 37°C in 5% CO₂. They were recently authenticated (Russ et al., 2012 (link)) and checked for contamination.