Cholera toxin

MCF-10A (non-tumorigenic epithelial cell line), MCF7, and A594 cells were purchased from ATCC. MCF-10A cells were cultured in Mammary Epithelial Cell Growth Medium (Takara) supplemented with 0.1 μg/ml cholera toxin (Sigma). MCF7 cells were cultured in MEM (Gibco) supplemented with 10% FBS (Gibco), 1% sodium pyruvate (Gibco), 1% glutamine (Gibco), and 0.1% insulin (Cell Science Technology). A594 cells were cultured in DMEM supplemented with 10% FBS. MCF-10A and TP53 (^−/−) cells were purchased from horizon. MCF-10A and TP53 (^−/−) cells were cultured in DMEM/F-12 medium (Gibco) containing 2.5 mM L-glutamine and 15 mM HEPES (Gibco), supplemented with 5% horse serum (Gibco), 0.5 μg/ml hydrocortisone (Sigma), 10 μg/ml human insulin (R&D System), 20 ng/ml hEGF (Sigma), and 0.1 μg/ml cholera toxin (Sigma). MDA-MB-231, T47D, U2OS, and 293T cells were generously provided by the Japanese Foundation for Cancer Research. MDA-MB-231 cells were cultured in DMEM/F-12 (Sigma). T47D cells were cultured in RPMI 1640 (Sigma) supplemented with 10% FBS and 0.2 unit/ml insulin. U2OS and 293T cells were cultured in DMEM supplemented with 10% FBS. Cells were cultured at 37 °C with 5% CO₂. All cell lines were examined to be mycoplasma negative before experiments.

BPECs and HMECs were obtained from mammoplasty specimens of disease-free patients, and were propagated according to conditions described by Ince and colleagues [1 (link)]. All necessary ethical approvals and consents were obtained for the collection and use of tissue samples for research purposes. BPECs were cultured in BD Primaria surface (BD Bioscience) using WIT-P-NC medium (initially supplied by Stemgent, Cambridge, MA, USA, ref 00–0051, and more recently by Cellaria, Boston, MA, USA, ref CM-0104) supplemented with 100 ng/ml cholera toxin (Sigma-Aldrich, Tres Cantos, Spain, ref C8052). HMECs were cultured in standard plates with serum-free MEpiCM (ScienCell, Research Laboratories, Carlsbad, CA, USA). BPECs and HMECs were grown at 37 °C and in 5 % CO₂. The number of accumulated population doublings (PDs) per passage was determined using the equation: $\mathrm{P}\mathrm{D}={\mathrm{P}\mathrm{D}}_{\mathrm{initial}}+log\left(N\mathrm{viable}\mathrm{cells}\mathrm{harvested}/N\mathrm{viable}\mathrm{cells}\mathrm{plated}\right)/log2\text{.}$
BPECs expressing hTERT (04BPEC-hTERT, 05BPEC-hTERT and 12BPEC-hTERT) were generated by lentiviral transduction with the hTERT gene at a pre-stasis PD (04BPECs at PD 3, 05BPECs at PD 4 and 12BPECs at PD 10) in the presence of 4 mg/ml Polybrene (Sigma-Aldrich). For propagation of BPEC-hTERT cell lines, WIT-T-NC medium (Cellaria, ref C10103) was supplemented with 25 ng/ml cholera toxin (Sigma-Aldrich, ref C8052).

Experiments were performed on the MCF-10A cell line purchased from ATCC (Manassas, VA, USA). Cells were cultured in 2D in DMEM/F12 (Life Technologies, Carlsbad, CA, USA) containing 5% horse serum (New Zealand origin, Life Technologies, Carlsbad, CA, USA), 20 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin (all Sigma-Aldrich). Acini from MCF-10A were cultured as previously described [30 (link)]. In brief, single cells were seeded onto a Geltrex (ThermoFisher Scientific, Waltham, MA, USA) bed and cultivated for nine days in EGF-supplemented assay medium (DMEM/F12 Glutamax, 2% horse serum, 5 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 1 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin), changing the medium every three days. At day nine, EGF was eliminated from the assay medium for further cultivation, changing the medium every three days [30 (link)], while acini cultivation did not exceed 21 days.