Nfat1

The lung adenocarcinoma cell line, PC-9, and its gefitinib-resistant sub-cell line, PC-9/GR possessing the T790M mutation, were gifted by Dr. Jin Kyung, Rho [13 (link)]. Cells were cultured in RPMI-1640 (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C in 5% CO₂. To determine the effects of [Ca²⁺]e restriction, the culture medium was exchanged with RPMI-1640 without CaCl₂ (MyBioSource, CA, USA) and 1 mM CaCl₂ was added. Cyclopiazonic acid was purchased from Enzo Life Sciences (Farmingdale, NY, USA). The mouse monoclonal antibodies against nuclear factor of activated T cell (NFAT1; cat # sc-7296), Orai1 (cat # sc-377281), Bcl-2 (cat # sc-509), and Bax (cat # sc-20067) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). U-compounds (U73122 and U73343) were obtained from Sigma Aldrich (St. Louis, MO, USA). Human EGF, gefitinib, and rabbit polyclonal antibodies against poly (ADP-ribose) polymerase-1 (PARP; cat #9542), stromal interaction molecule 1 (STIM1; cat #5668), sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2; cat #4388), and β-actin (cat #4967) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Western blotting was performed as previously described [20 (link)]. The blots were incubated overnight with primary antibodies targeting β-actin (Santa Cruz), Piezo1 (Poteintech), NFAT1 (Santa Cruz), NFAT2 (Santa Cruz), NFAT3 (Santa Cruz), NFAT4 (Santa Cruz), and TBP (Abcam) at 4 °C with constant shaking. After incubating with HRP-labeled secondary antibodies (Cell Signaling) at room temperature for 2 h, the protein bands were detected using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific).