Au565

Human kidney embryo cell line HEK293 (WT-HEK), breast cancer cell line MDA-MB-231 (WT-231), AU565, and T47D were obtained from ATCC^® (Manassas, VA, USA). The murine TRPM7 gene (GenBankTM accession number AF376052) overexpressing HEK293 clone (HEK-M7) was a gift from Dr. Loren Runnels, Robert Wood Johnson Medical School (Piscataway, NJ, USA) [32 (link)]. TRPM7 knock-out MDA-MB-231 cell line (KO-231) was obtained commercially from GenScript (Piscataway, NJ, USA) through GenCRISPRTM Technology and the knockout confirmed by genetic sequencing. The genetic sequencing shows a frameshift mutation at the TRPM7 gene (evidence can be provided upon reasonable request). HEK293 and MDA-MB-231 were cultured in Dulbecco’s Modified Eagle Media (DMEM) with 10% Fetal bovine serum (FBS), 10 mM piperazineethanesulfonic acid (HEPES), and 2% pen/strep. AU565 and T47D were cultured in Roswell Park Memorial Institute (RPMI)-1640 with 10% FBS, 10 mM, HEPES, and 2% pen/strep. All cell lines were cultured at 37 °C in a 5% CO₂ humidified incubator and the medium was exchanged once per two days.

MCF-10A cells and their derivatives MCF-ErbB2 and MCF-MekDD cells were provided by M. Reginato (Drexel University, USA) [16 (link)]. MCF10A cells were authenticated as published [11 (link)]. Lack of mycoplasma contamination in all cells was established as published [11 (link)]. BT-474 (American Type Culture Collection (ATCC), Manassas, VA, USA), BT474TR and BT474T cells were cultured as published [11 (link)]. Generation of BT-474TR cells is published [17 (link)]. To generate tumorigenic BT474T cells, BT474 cells were implanted orthotopically into the mammary fat pad of severe combined immunodeficiency mouse, and resulting tumors were serially passaged into new hosts over a three-year period [18 (link)]. AU-565 (ATCC), SKBR3 (ATCC) and 293 T cells (provided by A. Stadnyk, Dalhousie University) were cultured as published [11 (link)]. To detach the cells from the ECM, they were plated in suspension culture above a layer of 1% sea plaque agarose polymerized in respective culture medium not containing any additional ingredients.

We selected several breast cancer cell lines that were isolated from primary invasive ductal breast carcinomas (IDC; BT-483 (HTB-121) and BT-474 (HTB-20), American Type Culture Collection, ATCC, Manassas, VA) [36 (link)], metastatic pleural or pericardiac effusions (AC; MCF-7 (HTB-22), MDA-MB-453 (HTB-131), and AU-565 (CRL-2351), ATCC, Manassas, VA) [37 (link)-39 (link)], or other metastatic deposits (MC; MDA-MB-231 (HTB-26), ATCC, Manassas, VA) [40 (link)]. Additionally, a continuous cell line (HBL-100, denoted as N; Cat. No. HTB-124; ATCC, Manassas, VA) obtained from a primary culture of cells derived from an early lactation sample of human milk and a normal human breast epithelial cell line (MCF-10A, denoted as F; Cat. No. CRL-10317; ATCC, Manassas, VA) were used as controls. MCF-10A cells were maintained in complete MCF-10A culture medium composed of a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with 100 ng/mL cholera enterotoxin, 10 μg/mL insulin, 0.5 μg/mL hydrocortisol, and 20 ng/mL epidermal growth factor (Life Technologies, Rockville, MD, USA). MCF-7, MDA-MB-231, HBL-100, and MDA-MB-453 cells were maintained in DMEM; AU-565, BT-474 and BT-483 cells were cultured in RPMI-1640.

The following cell lines were obtained from the American Type Culture Collection (ATCC): i) TNBC lines MDA-MB-157, MDA-MB-436, MDA-MB-468, HCC70, BT-549, ii) ER+ /HER2/neu negative cell lines T-47D, ZR-75-1, MCF-7, MDA-MB-415, HCC1428, BT-483, iii) ER⁺/ Her2/neu⁺ cell lines BT474, and MDA-MB-361 and iv) ER-/Her2/neu⁺ cell lines AU565, HCC1954, and SKBR3. All cells except HS578T were cultured in DMEM-F12 containing 10% FBS. The TNBC line, HS578T also obtained from ATCC, was grown in DMEM with reduced NaHCO₃ (ATCC) containing 0.1 mM insulin and 10% FBS. The ER⁺/ Her2/neu-overexpressing MCF7 (MCF7-Her18) cells were a kind gift from Dr. Elizabeth Mittendorf. All the cell lines used in here were strictly with in ten passages after buying from ATCC and thus were not authenticated again.

The cell lines used in this study: AU565, BT20, BT474, BT483, BT549, HCC1143, HCC1500, HCC1569, HCC1937, HCC1954, HCC38, HCC70, Hs578T, MDA-MB-134, MDA-MB-175, MDA-MB-361, MDA-MB-436, MDA-MB-453, MDA-MB-468, SKBR3, and ZR751 were obtained from ATCC, Rockville, MD, USA. MCF-7 cells were obtained from Michigan Cancer Foundation, Detroit, MI, USA. The cell lines HBL100, MDA-MB-157, MDA-MB-231 and T47D were obtained from EG&G Mason Research Institute, Worcester, MA, USA.
The cell lines AU565, BT20, BT474, BT549, HBL100, Hs578T, MCF-7, MDA-MB-134, MDA-MB-157, MDA-MB-175, MDA-MB-231, MDA-MB-361, MDA-MB-436, MDA-MB-453, MDA-MB-468, SKBR3, T47D and ZR571 were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 6 mM L-glutamine, 20 mM HEPES and 10 μg/ml human insulin (CSL-Novo, North Rocks, NSW, Australia). The remaining cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 6 mM L-glutamine, 1 mM sodium pyruvate and 20 mM HEPES. The MYC over-expressing MCF7 cells have been previously described
[11 (link),27 (link)] and were cultured in the same conditions as the parental cells.
The CDK4/6 inhibitor PD0332991 was purchased from Selleck Chemicals (Houston, TX, USA), CDK2 inhibitor SNS-032 from Symansis (Auckland, New Zealand) and CDK1 inhibitors, RO-3306 and CGP74514A, from Calbiochem (San Diego, CA, USA).