Megm mammary epithelial cell growth medium

MCF7 (HTB-22 from ATCC, Manassas, VA, USA), a model of human sporadic breast cancer cell line, luminal A, were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, Saint Louis, MO, USA, implemented with 100 mg/mL streptomycin and 100 U/mL penicillin (Sigma Aldrich) and 10% (w/v) fetal bovine serum (FBS) (Sigma Aldrich). MCF10 are a model of immortalized mammary epithelial cells (CRL-10317 from ATCC, Manassas, VA, USA). Cells were grown in MEGM, Mammary Epithelial Cell Growth Medium (Lonza, Walkersville, MD, USA), implemented with 20 ng/mL epidermal growth factor (Lonza),10 μg/mL insulin (Lonza), 0.5 μg/mL hydrocortisone (Lonza), and 100 ng/mL cholera toxin (Sigma Aldrich). The HCC1937 (CRL-2336 from ATCC, Manassas, VA, USA) are a model for hereditary breast cancer cell line that has a homozygous mutation for the BRCA1 5382C terminal; The HCC193 are a model of triple negative breast cancer. The culture medium for HCC1937 was Roswell Park Memorial Institute (RPMI) (ATCC, Manassas, VA, USA) implemented with 20% (w/v) fetal bovine serum (FBS) (Sigma Aldrich), 100 mg/mL streptomycin, and 100 U/mL penicillin (Sigma Aldrich).

MDA-MB-231 and SUM159 cells were kind gifts from Dr. Jun-Lin Guan and Dr. Susan Waltz, respectively. Cells were authenticated using short tandem repeat analysis (Laragen, Inc.). MDA-MB-231 and HEK293T cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. SUM159 and HCC1806 cells were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. MCF10A cells (ATCC) were cultured in MEGM Mammary Epithelial Cell Growth Medium (CC-3150, Lonza). HEK293T cells used for lentivirus production were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were kept in a 37°C, 5% CO₂ humidified incubator and were routinely tested for mycoplasma contamination using Mycoplasma PCR Detection Kit (Applied Biological Materials) and only mycoplasma free cells were used in experiments. An MDA-MB-231 CRISPR-cas9 single cell clone was isolated by plating lentiCas9-Blast (52962, Addgene) transduced cells into single cells in 96-well plates and selecting with 5 μg/ml of blasticidin.

E14 mouse embryonic stem cells (mESCs) were cultured in high glucose DMEM media, supplemented with 15% ES grade fetal bovine serum (FBS; Biowest S182S‐500, Batch S10397S182), sodium pyruvate, gentamycin (Gibco 15710‐064), non‐essential amino acids (NEAA; Gibco 11140‐050), glutamax (Gibco 35050‐061), β‐mercaptoethanol (Gibco 21985023), and 1,000 U/ml LIF (ESGRO 1106). The mESCs were grown on 0.1% gelatin‐coated plastic and passaged every 2 days, with daily media changes, and for a maximum of 12 passages. mESCs were differentiated with 0.5 μM RA for 48 h, unless specified otherwise. HCT116 Myc‐Luc reporter cells were purchased from Amsbio, BPS Bioscience (#60520). MDA‐MB‐231 and HS578T breast cancer cell lines were gifts from Wai Leong TAM. The above were cultured in high glucose DMEM with sodium pyruvate, supplemented with 10% FBS (Biowest S181B‐500), 1% penicillin/streptomycin (Gibco 15140‐122), and 1% glutamax (Gibco 35050‐061). The HCT116 Myc‐Luc reporter line is additionally cultured under selection with 500 μg/ml geneticin. Human mammary epithelial cells (HMECs) 1–3 were a gift from Mathijs Voorhoeve. The HMECs were maintained in MEGM™ Mammary Epithelial Cell Growth Medium (Lonza MEGM BulletKit; CC‐3151 and CC‐4136).

In vitro analyses were performed on a triple negative breast cancer cell line MDA-MB-231 and healthy mammary gland cell line MCF10A. Both cell lines were grown at 37°C, 5% CO₂ and 80% confluence. Maintenance medium for MDA-MB-231 cells was DMEM-GlutaMax (4.5gr D-Glucose/Liter, ThermoFisher) enriched with Fetal Bovine Serum (FBS-10%), Non Essential Amino Acids (NEAA-1%) and Streptomycin-Penicillin (Strepto/Pen). Maintenance medium for MCF10A cells was DMEM F:12 enriched with MEGM™ Mammary Epithelial Cell Growth Medium (Lonza), 1gr/L Glucose and the 10% FBS. For STS experiments DMEM glucose-free (ThermoFisher) enriched with 0.5 gr/L Glucose, 1% FBS, 1% NEAA, 1% Strepto/Pen and 25 mM Hepes was used.
Cells were treated with 1 μM Doxorubicin for 24 h and STS conditions were maintained at different time points (24, 48 and 72 hours).

MCF-10, MCF-7 and HCC1937 cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). Human breast cancer cell line MCF7 was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, Saint Louis, MO, USA) supplemented with 10% (w/v) fetal bovine serum (FBS) (Sigma Aldrich), 100 mg/mL streptomycin and 100 U/mL penicillin (Sigma Aldrich). MCF10 mammary epithelial cell line was grown in MEGM Mammary Epithelial Cell Growth Medium (Lonza, Walkersville, MD, USA) supplemented with 20 ng/mL epidermal growth factor (Lonza), 0.5 µg/mL hydrocortisone (Lonza), 100 ng/mL cholera toxin (Sigma Aldrich) and 10 µg/mL insulin (Lonza). HCC1937 cell line was homozygous for the BRCA1 5382C mutation and was used as a model of hereditary breast cancer. HCC1937 cells (ATCC) were grown in RPMI medium (ATCC) supplemented with 20% (w/v) fetal bovine serum (FBS) (Sigma Aldrich), 100 mg/mL streptomycin and 100 U/mL penicillin (Sigma Aldrich).
All cell lines were cultured at a constant temperature of 37 °C in a 5% carbon dioxide (CO₂) humidified atmosphere.

The human HeLa-S3 immortalized cell line (Catalog No. 30-2004) and human SK-BR-3 cell line (Catalog No. 30-2007) were grown at 37 °C with 5% CO₂ in high-glucose DMEM (Thermo Scientific, 11965084) containing 10% (v/v) fetal bovine serum (HyClone, SV30087.02) and 100 U/mL Penicillin Streptomycin (Thermo Scientific, 15140163). The human MCF10A cell line (Catalog No. CC-3150) was grown at 37 °C with 5% CO₂ in MEGM Mammary Epithelial Cell Growth Medium (Lonza, CC-3150) containing supplements required for growth. The human MDA-MB-157 cell line (Catalog No. 30-2008) was grown at 37 ℃ without CO₂ equilibration in Leibovitz’s L-15 Medium (Thermo Scientific, 11415-064) supplemented with 10% (v/v) fetal bovine serum and 100 U/mL Penicillin Streptomycin. The human HCC1395 cell line was cultured in RPMI 1640 Medium (Thermo Scientific, 11875093) containing 10% (v/v) fetal bovine serum and 100 U/mL Penicillin Streptomycin at 37 °C with 5% CO₂.
Mouse ESCs were cultured as previous described [59 (link)]. Briefly, mESCs were grown on gelatin-coated plates in DMEM supplemented with 10% FBS, penicillin/streptomycin, 5 μM 2-Mercaptoethanol, 2 mM L-glutamine, non-essential amino-acids and 10 ng/mL recombinant Leukaemia-Inhibitory Factor (LIF).