Figure 7.

The inhibition of Orai1 channel abolished PL-induced Ca²⁺ entry. A. PL-induced Ca²⁺ entry was completely abolished in the presence of 1 µM Pyr6 (30 min preincubation). PL was added at 20% v/v. Data are means ± s.e.m. of [Ca²⁺]_i traces recorded in different cells. Number of cells: PL alone: 40 cells from 3 experiments; PL + Pyr6 1 µM: 50 cells from 3 experiments. B. Mean ± s.e.m. of the Ca²⁺ response to 20% PL recorded at the peak and at the plateau under the designated treatments. Data are means ± s.e.m. of [Ca²⁺]_i measured by confocal imaging at peak maxima. Number of cells: PL alone: 40 cells from 3 experiments; PL + Pyr6 1 µM: 50 cells from 3 experiments. Asterisks on bars indicate statistical differences determined by two-way ANOVA with Bonferroni’s correction (p < 0.001). C. Using the Mn²⁺-quenching technique, the resting Ca²⁺ entry in bEND5 cells was evaluated. First, the extracellular medium was replaced with a 0Ca²⁺ solution and then, to cause an immediate decay in Fura-2 fluorescence, 200 µM Mn²⁺ was added. PL 20% treatment allowed an evident decay of fluorescence, while pre-incubating the cells with Pyr6 strongly prevented this decay. CTRL indicates cells incubated in 0Ca²⁺ solution. D. The quenching rate of Fura-2 fluorescence induced by Mn²⁺ addition was calculated as the slope of a linear regression. Different asterisks on bars indicate statistical differences (*** p < 0.001; ** p < 0.005).