Mepicm

MCF-10A cells, the human normal breast cells, were purchased from Zhongqiaoxinzhou Biotech (Shanghai, China) and cultured in Mammary Epithelial Cell Medium (MEpiCM, ScienCell, Research Laboratories, Carlsbad, CA, USA). BC cell lines including MDA-MB-231, MCF-7, HCC-1937, SKBR3, MDA-MB-468 were purchased from the Chinese Academy of Sciences (Shanghai) and cultured in Dulbecco's Modified Eagle’s Medium (DMEM) (Gibco, USA) adding 10% Fetal Bovine Serum (FBS) (Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Enpromise, China). The cell cultural condition of the cell incubator was set at 37°C with 5% CO2. 2 μg/ml actinomycin D (Millipore, Billerica, MA, USA) was added to the DMEM to inhibit transcription of MDA-MB-231 and MDA-MB-468 cells for 0 h, 8 h, 16 h, and 24 h.

All cancer cell lines were purchased from the American Type Culture Collection. Media used for the maintenance of these cell lines are listed as follows: MDA-MB-231, Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, 12430-054); LNCaP and PC-3, Roswell Park Memorial Institute (RPMI) 1640 medium (RPMI 1640, Life Technologies, 22400-089); All media was supplemented with 10% fetal bovine serum (FBS, Gibco, 16000-044) and penicillin-streptomycin unless otherwise indicated. Mammary epithelial cells MCF-10A were also supplied by the American Type Culture Collection. Mammary Epithelial Cell Medium (MEpiCM, Sciencell, 7611) was used for incubation, consisting of 500 ml of basal medium, 5 ml of mammary epithelial cell growth supplement (MEpiCGS, Sciencell, 7652) and 5 ml of penicillin/streptomycin solution (P/S, Sciencell, 0503). Cells were incubated at 37 °C in 5% CO₂ in a humidified incubator.

Breast cancer cell lines including MDA-MB-231, MDA-MB-468, SKBR3, T47D, BT474, MCF7, and MCF10A which were primarily from ATCC and stored in our lab. Cells were maintained in DMEM/F12, or L15 medium containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. MCF10A cells were cultured in a mammary epithelial cell medium (MEpiCM, ScienCell). CAFs were maintained in DMEM/F12 (Hyclone, USA) containing 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. All the cell lines were cultured in a humidified atmosphere of 5% CO₂ at 37 °C. For hypoxia, the cells were incubated in a chamber with 1% O₂.

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).