Cholera toxin

The human breast cancer cell lines MCF-7 and MDA-MB-231, human mammary epithelial cells with integrated SV40 gene (HBL-100) as well as non-malignant mammary epithelial cell line MCF-10A were obtained from ATCC (Manassas, VA, United States). All the above cell lines have been identified by short tandem repeat analysis. RPMI-1640 medium (Gibco Life Technologies, Lofer, Austria) for MCF-7 cells and Dulbecco's Modified Eagle medium (DMEM, Gibco Life Technologies, Lofer, Austria) for MDA-MB-231 cells and HBL-100 cells were applied in exception to 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin (Gibco). And MCF-10A cells were maintained in DMEM/F12 medium (Gibco) supplemented with 5% horse serum (HyClone, Logan, Utah, United States), 1% penicillin and streptomycin, 20 ng/ml recombinant human epidermal growth factor (BD Bioscience, Bedford, MA, United States), 0.5 μg/ml hydrocortisone (STEMCELL Technologies, Vancouver, Canada), 100 ng/ml cholera toxin (MACGENE, Beijing, China) and 10 μg/ml insulin (Sigma, St. Louis, MO, United States). DMEM/F12 medium for the CSCs derived from MDA-MB-231 and MCF-7 cells were employed in addition to B27 (Invitrogen, Carlsbad, CA, United States), 5 μg/ml of insulin, 20 ng/ml of hEGF, 1% penicillin and streptomycin and 0.4% BSA (Sigma) at 37°C with 5% CO₂.

The human normal epithelial breast cell line MCF-10A and BC cell lines MCF-7, BT-549, MDA-MB-231, and MDA-MB-468 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MCF-7, MDA-MB-231, and MDA-MB-468 cells were cultured in DMEM (Gibco, CA, United States) and BT-549 cells were cultured in RPMI-1640 medium (Gibco, CA, United States) supplemented with 10% fetal bovine serum (Australia Origin, Gibco, Carlsbad, CA, United States) and 1% penicillin/streptomycin (Solarbio, Beijing, China), respectively. Meanwhile, MCF-10A cells were cultured in DMEM/F12 (MACGENE, Beijing, China) with 5% HS (horse serum; EVERY GREN, Hangzhou, China), 10 μg/ml insulin (MACGENE, Beijing, China), 20 ng/ml EGF (MACGENE, Beijing, China), 100 ng/ml cholera toxin (MACGENE, Beijing, China), and 0.5 μg/ml hydrocortisone (MACGENE, Beijing, China). The cells were grown in a humidified atmosphere of 5% CO₂ at 37°C and not contaminated by mycoplasma.

The HEK-293T cell line, human mammary epithelial cell line MCF-10 A, and human breast cancer (BC) cell lines MDA-MB-231, MDA-MB-468, MCF-7, and BT549 were purchased from the Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences (Shanghai, China). Cell lines were maintained under standard media and conditions. MDA-MB-231, MDA-MB-468, MCF-7, and HEK293T were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Rockville, MD, USA) and 1% antibiotic solution. The culture medium for BT549 was Roswell Park Memorial Institute (RPMI, Gibco)1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% FBS and 1% antibiotic solution. MCF-10 A was cultured in DMEM/F12 (Macgene, Beijing, China) medium supplemented with 5% horse serum, 10 μg/ml insulin (Macgene, Beijing, China), 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin (Macgene, Beijing, China), and 0.5 μg/ml hydrocortisone (Macgene, Beijing, China). All cell lines were cultured at 37° C, 5% CO₂ in a humidified cell culture incubator.