Fig. 2: SOCE mediates Ca²⁺ entry in crystal-internalized HK2 cells.

Mixed crystals were introduced into HK2 cells in a concentration-dependent manner (crystal concentrations: 8 µg/mL, 24 µg/mL, and 80 µg/mL) for 24 h. Following mixed crystal internalization, effects on [Ca²⁺]_i mobilization on CaSR-activated HK2 cells were measured by Ca²⁺ imaging. Representative bar diagrams depict a peak Ca²⁺ entry and, b peak [Ca²⁺]_i release. To delineate the pathway of Ca²⁺ entry, c control (noncrystal); d CaP; and e Mixed crystals were introduced into HK2 cells; following crystal internalization, cells were left untreated (control) or treated with SOCE blocker (Pyr6) or ROCE blocker (Pyr10). Mean fluorescent traces of Fura-2 loaded HK2 cells bathed in Ca²⁺-free solution and then 2.0 mM Ca²⁺ and neomycin CaSR activation were obtained for c Control; d CaP; and e Mixed crystals. Inset bar diagrams in c–e represent peak [Ca²⁺]_i rise. HK2 cells were incubated overnight with (c) control, CaP crystals (d), and mixed crystals (e). Cells were bathed in Ca²⁺-free solution and incubated for 5 minutes with 3 µM of Pyr6 or 10, and 50 µM neomycin was applied, followed by 2.0 mM Ca²⁺. Two-tailed t-test was used for statistical comparison in a and b. Statistically significant differences are indicated (mean ± SEM) from four different experiments. Levels of significance are indicated as *p < 0.05; **p < 0.01 as shown in the bar diagrams