Fig. 3: Evaluation of the effect of XE-991 on the increase in cytoplasmic and mitochondrial Ca²⁺ levels induced by GA in primary rat cortical neurons.

A, C The bar plots depict average data of the cytoplasmic (A) and mitochondrial (C) basal Ca²⁺ levels. The fluorescence intensity is transformed into calibrated [Ca²⁺] by applying the following equation: [Ca²⁺] = Kd (F − F_min) / (F_max − F) where Kd is the Ca²⁺ dissociation constant of the indicator (Kd Fluo-4-AM = 345 nM; Kd Rhod-2-AM = 570 nM); F_min and F_max represent the intensities of fluorescence at zero and saturating [Ca²⁺], respectively; and F represents the intensity of fluorescence at any given time. In each experiment, [Ca²⁺] was expressed as control percentage. B, D Representative images of the basal Ca²⁺ levels within cytoplasm (B) and mitochondria (D). Scale bar 50 µm. Differences were evaluated by one-way ANOVA followed by Dunnett’s post hoc test. A F (3, 10) = 11.67. For each experimental group, the statistical analysis was performed by using the basal values derived from three independent experiments, and 100–150 cells were recorded for each session. *Significant versus all groups (p < 0.01 versus Ctl, p < 0.001 versus XE-991, and p < 0.05 versus XE-991 + GA). C F (3, 15) = 8.091. For each experimental group, the statistical analysis was performed by using the basal values derived from at least four independent experiments, and 100–150 cells were recorded for each session. *Significant versus all groups (p < 0.01 versus control groups and p < 0.05 versus XE-991 + GA).