Bt 20

MCF7, BT549, MDAMB231, MDAMB468, Hs578T, SUM159PT, BT549, and BT20 breast cancer cell lines were obtained from ATCC (Manassas, VA, USA). MCF7 cells were grown in DMEM medium, the other cell lines were grown in RPMI medium, with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL). Cells were plated 24 h before treatment with different drugs at the indicated concentrations. Recombinant human EGF, heregulin (HRG), IGF1, angiotensin II (Ang II), and angiotensin 1–7 (Ang 1–7) were obtained from Sigma Chemical Co (St. Louis, MO, USA). Recombinant HGF and FGF were from Abcam. Recombinant PDGF-BB was from Gibco. Trastuzumab, gifitinib, lapatinib, sunitinib, OSI-906, and doxorubicin were obtained from Selleck Chemicals. The PRCP inhibitor (PRCP-7414, referred to as PRCPi in text) was from Calbiochem (catalog number 504044).

BT-20, HCC70, MDA-MB-468, BT-549, MDA-MB-231, MDA-MB-436, and MCF-10A cells were purchased from the ATCC (Manassas, VA, USA). SUM159 cells were purchased from Astrerand (now BioIVT, Westbury, NY, USA) and iMEC cells were provided by Dr. Elizabeth Alli in the Department of Cancer Biology at Wake Forest School of Medicine. Cell lines were expanded, and low passage stocks were stored in liquid nitrogen and maintained by the Wake Forest Comprehensive Cancer Center Cell Engineering Shared Resource. Cell lines and growth media are listed in Table 1.
All cells were verified to be free from mycoplasma contamination by routine testing using the MycoAlert Mycoplasma Detection Kit (Lonza, Morristown, NJ, USA). Cells were passaged and medium was changed twice weekly. Cell monolayers were grown on tissue culture treated plastics purchased from Corning Life Sciences (Corning, NY, USA) or on glass coverslips (Warner Instruments Corporation, Hamden, CT, USA). For live fluorescence imaging studies, cells were grown in 8-well chamber slides (EMD Millipore, Burlington, MA, USA). Cells were maintained in culture for no longer than 4 months before new cultures were established from low-passage frozen stocks.

MCF10A cell line was provided by Potier-Cartereau (Université François Rabelais, Tours, France). MCF7 and the triple-negative breast cancer cell lines—MDA-MB-231, MDA-MB-468, and BT20, were collected from ATCC^® (Manassas, VA, USA). Cells were cultured following the manufacturer’s instructions or the protocols described elsewhere [25 (link)]. MCF7 and the TNBC cells were cultured at 37 °C with 5% CO₂ in DMEM medium supplemented with FBS and penicillin/streptomycin; meanwhile, MCF10A cells were cultured in DMEM F-12 supplemented with horse serum, insulin, hydrocortisone, EGF, cholera toxin, and penicillin/streptomycin. Cell transfection was achieved by cell permeabilization with DharmaFECT transfection reagent, and subsequently, cells were incubated for 48 h either with 1–3 μg/mL of esiRNAs against STIM1 and STIM2 or siRNA. Alternatively, we used 2 μg/mL of scrambled plasmid, shPGRMC1, shOrai1, and shTRPC1 plasmids. In order to ascertain the efficiency of silencing protocols, Western blotting using the specific antibodies was done, as described below. Finally, cell transfection was done using the YFP-NFAT1-reporter overexpression plasmid (2 μg/mL), which was evaluated upon 72 h of transfection in order to ascertain NFAT1 translocation to the nucleus due to Ca²⁺ entry activation [80 (link)].