Cell Culture and Transfection Protocols

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)].