Thapsigargin tg

The calcium assay was performed as described previously.^{50 (link)} The cells were plated onto gelatin-coated 30 mm plates 1 day before the experiments. The cells were washed with Tyrode's solution and incubated with 4 μM Fluo-4 AM (Invitrogen) or Fura-4 AM (Invitrogen) for 30 min at room temperature. The cells were then washed with Tyrode's solution. To measure store-operated Ca²⁺ influx, 4 mM EGTA and 4 mM thapsigargin (TG) (Abcam) were added to deplete internal calcium stores. Ca²⁺ influx was induced by the subsequent addition of 2 mM Ca²⁺ (free) after store depletion. The change in calcium fluorescence was monitored using a confocal fluorescence imaging microscope (LSM710, ZEISS), which was performed in Professor Heping Cheng’s laboratory at the Institute of Molecular Medicine, Peking University.

2-Amino-ethoxydiphenyl borate (2-APB, 100 µM), Nifedipine (10 µM), LaCl₃ (100 µM), Cytochalasin D (1 µM), Blebbistatin (20 µM), Nocodazole (1 µM), ML-7 (20 µM), the ROCK inhibitor Y27632 (20 µM), and phalloidin-TRITC were purchased from Sigma-Aldrich. Thapsigargin (TG, 10 µM) was purchased from Abcam, U73122 (10 µM) from MedChemExpress, and fibronectin from Corning. ITGB1 (Beta1) siRNA (Integrin siRNA, 30 nM) was purchased from Thermo Fisher Scientific.

Primary airway smooth muscle (ASM) cells were originated from 6–8-week-old Sprague–Dawley rats, as described previously (approved by the Ethics Committee of Changzhou University on Studies Ethics, Grant No. NSFC 11532003) (Wang et al., 2016 (link)). ASM cells were cultured in low-glucose DMEM (Invitrogen) supplemented with 10% FBS and penicillin/streptomycin antibiotics. The cells were maintained in the humidified culture incubator, containing 5% CO2 at 37°C. The cells applied in the experiments were generally within 10 passages during regular culture.
Matrigel was purchased from BD Biotechnology, and type I collagen was from Advanced Biomatrix. The chemical reagents 2-amino-ethoxydiphenyl borate (2-APB, #D9754-10G), nifedipine (#N7634-1G), cytochalasin D (1 µM), blebbistatin (20 µM), and ML-7 (20 µM) were purchased from Sigma-Aldrich, and thapsigargin (TG, #ab 120286) from Abcam. ON-TARGETplus SMARTpool N-cadherin siRNA (N-cadherin siRNA, #M-091851-01-0005) was purchased from Horizon Discovery. Integrin β1 (ITGB1) siRNA (#AM16708) was purchased from Thermo Fisher Scientific.