Humec supplement

Breast fibroblast cells were obtained and used as previously described [21 (link)]. NBFs (normal breast fibroblasts) are cultures obtained from tissues derived from reduction mammoplasty, CAFs (cancer-associated fibroblasts), and TCFs (tumor counterpart fibroblasts) are from histologically normal parts of tumors. Fibroblasts were cultured in M199 medium (Gibco, Grand Island, NY, USA ) and Ham’s F12 (Gibco) mixed 1:1 and supplemented with 20% Heat Inactivated Fetal Bovine Serum (FBS) (Gibco) and 1% Antibiotic-Antimycotic 100X (Gibco). Breast cancer cells MDA-MB-231 and MCF-7 were obtained from ATCC and were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 1% Antibiotic-Antimycotic, while Human Mammary Luminal Epithelial (HMLE) cells were a generous gift from Dr. Hazem Ghebeh (KFSHRC) and were maintained in DMEM/F12 (Gibco) supplemented with 0.5% Bovine Pituitary Extract (Gibco) and 1% HuMEC supplement (Gibco) in addition to 2% FBS and 1% Antibiotic-Antimycotic. Cells were maintained at 37°C in humidified incubator with 5% CO₂. For long storage, cells were kept in freezing media (Gibco). Recombinant IL-6 was purchased from GenWay Biotech. Recombinant OPG was purchased from Sigma-Aldrich.

Mouse embryo fibroblasts (MEF) reconstituted for stable expression of DNA-PKcs (SCH8, DNA-PK +/+), wild type scid mice MEF (ScSV3, DNA-PK −/−) were kind gifts from Dr. Frederic Alt lab. HeLa cells, HCT-15 cells, BT-474 cells, HCC-1937 cells, MDA-MB-231 cells, SUM-52 cells, BT-549 cells, BT-20, HCT-116, Colo 205, H69, H249 and H526 were obtained from ATCC. MO59K cells and MO59J cells were kind gifts from Dr. Lees-Miller lab. SCH8 cells, ScSV3 cells, HeLa cells and MDA-MB-231 cells were grown and maintained in DMEM containing 10% fetal bovine serum, 2mM L-Glutamine, 100 units/ml of Streptomycin and 100 units/mL of Penicillin. HuMEC supplement (Invitrogen) was also used for SUM52 cells. BT474 cells, BT-549 cells, BT-20 cells, HCC-1937 cells, HCT-15 cells, HCT-116 cells, Colo 205 cells, H69 cells, H249 cells and H526 cells were maintained in RPMI containing 10% FBS and pen-strep. MO59K and MO59J cells were maintained in DMEM/F12 containing 10% FBS, 1mM MEM sodium pyruvate, 0.1mM NEAA and pen-strep. All cells were grown at 37°C and 5% CO₂ in a humidified cell culture incubator.

Human normal foreskin fibroblasts (BJ-5ta), purchased from American Type Culture Collection (ATCC, Manassas, USA), were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS). Human primary colonic epithelial cells (HCEC), purchased from Cell Biologics (Chicago, USA), were cultured in Epithelial Cell Medium /w Kit. Human embryonic kidney epithelial cells (HEK293), purchased from ATCC, were cultured in Eagle’s Minimum Essential Medium supplemented with 10% FBS. Human mammary epithelial cells (HMEC), purchased from Invitrogen were cultured in a HuMEC basal serum-free medium (Invitrogen) containing HuMEC supplement (Invitrogen). Human primary small intestinal epithelial cells (HSIEC), purchased from Cell Biologics were cultured in Epithelial Cell Medium /w Kit. Human lung fibroblasts (WI-38), purchased from ATCC, were cultured in Eagle’s Minimum Essential Medium supplemented with 10% FBS. All cell lines were incubated at 37 °C in a humidified atmosphere of 5% CO₂. Mycoplasma contamination was regularly monitored using a Mycoplasma PCR Detection kit (Applied Biological Materials Inc., Richmond, BC, Canada) and eradicated using BM-Cyclin (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer’s instructions.

Primary mammary normal epithelial cells (HUMEC, ATCC PCS-600-010) and MCF7 (ATCC HTB-22) were purchased from the American Type Culture Collection (ATCC, USA). The cells were cultured at 37 °C in a 5% CO₂ incubator. HUMEC were maintained in HUMEC Ready Medium containing HUMEC Basal Serum Free Medium, HUMEC Supplement, and bovine pituitary extract (Cat # 12752-010, Thermo Fisher Scientific, USA). MCF7 cells were cultured in DMEM (Cat # 10013CV, Corining, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (P/S, Thermo Fisher, USA).

The MCF-7/S0.5 (S05), MCF-7/182^R-6 (182^R-6), and MCF-7/TAM^R-1 (TAM^R-1) cell sublines were developed by Dr. Anne Lykkesfeldt (Breast Cancer Group, Cell Death and Metabolism, Danish Cancer Society Research Center, DK-2100, Copenhagen, Denmark). ICI 182,780 (Faslodex, fulvestrant) and tamoxifen-resistant sublines, 182^R-6 and TAM^R-1, respectively, are derived from S05 as described elsewhere [24 (link), 25 (link)]. These cell lines were cultured in a DMEM/F-12 medium with 2.5 mM L-Glutamine, without HEPES and phenol red (HyClone), and supplemented with 1% heat-inactivated fetal bovine serum (HyClone). Additionally, for 182^R-6 and TAM^R-1 sublines were regularly supplemented with 0.1 μM ICI 182,780 and 1 μM tamoxifen, respectively. Human mammary epithelial cells (HMEC) purchased from ThermoFisher Scientific (Cat# A10565) were cultured in a HuMEC basal serum-free medium (ThermoFisher Scientific) containing HuMEC supplement (ThermoFisher Scientific), 100 IU/mL penicillin, and 100 mg/mL streptomycin. All cell lines were incubated at 37 °C in a humidified atmosphere of 5% CO₂.