Breast Cancer Cell Line Culture and Manipulation

MCF-7 (ATCC^® HTB-22^™) human breast cancer cells were grown in DMEM, high glucose, pyruvate (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher). MCF-12A (ATCC^® CRL-10782^™) human breast epithelial cells were grown in DMEM/F12 (1:1) medium (Gibco), supplemented with 10% horse serum (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher), 2.5 mg/ml insulin (Invitrogen), 150 μg/ml cholera enterotoxin (Sigma-Aldrich), 2.5 mg/ml hydrocortisone (Sigma-Aldrich) and 20 ng/ml epidermal growth factor (Sigma-Aldrich). Bag-1 knockout MCF-7 cells were generated using CRISPR-Cas9 system [9 (link)]. Cells were maintained at 37°C, 5% CO₂ in a humidified incubator. 60–70% confluent cells were transfected with plasmids by using IN-fect in vitro transfection reagent (iNtRON Biotechnology) according to the manufacturer’s protocol. Cells were lysed 48 hours after plasmid transfection for protein purification or immunoblotting assays.