Measurement of intracellular calcium concentration

The intracellular calcium concentration was measured using a Ca²⁺-sensitive fluorescence indicator, cell-permeant Fluo-4/AM (Thermo Fisher Scientific). Cells were seeded onto 96-well plates (10,000 cells per well) or 24-well plates (30,000 cells per well) and treated 24 h later with drugs for various times. Then, cells were washed with Hank’s balanced salt solution (HBSS), incubated for 60 min at 37°C in dark in HBSS containing 3 µM Fluo-4/AM and 0.1% of Pluronic F-127, washed with HBSS, and incubated in dark for an additional 30 min at 37°C. Data were acquired using a microplate reader (CLARIOstar Plus; Bio-Rad) with the excitation and emission filter at 488 and 520 nm, respectively, or cells were imaged with Axio Observer Z1 (Carl Zeiss). For microplate experiments, protein concentration was further evaluated in each condition using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). A fluorescence intensity/protein concentration ratio was calculated for each well. Relative Fluo/protein concentration ratios were represented as a fold increase relative to untreated cells, which were arbitrarily assigned to 1.