Cytosolic Ca2+ levels were measured using a calcium ion fluorescent probe (Fluo-4 AM) assay kit. Cells were cultured in 96-well plates after treatment with 50 µM NPPB for 24 h at 37°C. The cells were washed three times with PBS, and Fluo-4 AM (final concentration of 1 µM) was added for 1 h at 37°C and washed three times with PBS. To ensure that the Fluo-4 AM was completely converted into Fluo-4 in the cells, another 30-min incubation at 37°C was necessary after washing. The cells were observed and photographed under a fluorescence microscope (magnification, ×400) at 488 nm.

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