Mcf10a

MCF10A (American Type Culture Collection), nontumorigenic human breast epithelial cells, MCF10A with anti-YAP short hairpin RNA (shRNA) (shYAP) (courtesy of G.D. Longmore, Washington University), and mutant MCF10A cells overexpressing ErbB2 (kindly provided by Dihua Yu), were cultured in DMEM/F12 (GE Healthcare Life Sciences), supplemented with 5% horse serum (Invitrogen), 20 ng/ml epidermal growth factor (EGF; Miltenyi Biotec), 0.5 mg/ml hydrocortisone (Sigma-Aldrich), 10 µg/ml insulin (Sigma-Aldrich), 100 ng/ml cholera toxin (Sigma-Aldrich), and 0.2 % Normocin (Invitrogen). Media was changed every 3 d while cells were in culture. For YAP depletion, the healthy MCF10A cell line was transfected using a lentiviral pFLRU vector containing anti-YAP shRNA, developed and verified in our previous work (Nasrollahi et al., 2017 (link)).

A total of 30 paired formalin-fixed paraffin-embedded human breast cancer tissue samples and adjacent non-cancerous tissue samples were obtained from the Department of Pathology of The Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) between May 2015 and December 2015. The patients were aged 35–79 years, with an average age of 59.0±12.7 years. No patients had received radiotherapy or chemotherapy prior to surgery. All the patients provided informed consent, and the study protocol was approved by the Ethics Committee of Xi'an Jiaotong University.
The human breast cancer cell lines, MCF-7 and MDA-MB-231, and the normal breast epithelial cell line MCF-10A, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 and MCF-7 cells were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% foetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). MCF-10A cells were maintained in DMEM/F12 medium (HyClone; GE Healthcare Life Sciences) with the addition of 10 µg/ml insulin, 20 ng/ml epidermal growth factor (Sigma-Aldrich; Merck KGaA) and 5% horse serum (Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured in a humidified incubator at 37°C with 5% CO₂.