Figure 4: Mitochondrial network distribution in basal and SOCE activated conditions.

(A) Confocal images of GDAP1-silenced neuroblastoma cells transfected with empty vector (mitochondria marked with α-βATPase, red signal), GDAP1 WT or mutant proteins (mitochondria marked with α-c-myc, red signal) and Orai1::CFP (green signal) in basal conditions and after ER-Ca²⁺ mobilization with 5 μM Tg in Ca²⁺-free medium. Cells were fixed after 10 min of vehicle or Tg treatment. Two images of each cell type and condition are shown; High resolution images of vehicle- (only in the case of dominant mutation) or Tg-treated cells are also shown on the right panel side. Bars indicate 10 μm. (B) Quantification of mitochondrial fluorescence distribution in basal conditions and after depletion of ER calcium stores with 5 μM Tg in Ca²⁺-free medium. Fluorescence distribution at the subplasmalemmal (SP) domains (defined as 2 μm underneath the plasma membrane) and the central zone (the space between the opposite subplasmalemmal domains) were calculated as indicated in Fig. S2. Fluorescence distribution in 7–9 cells per treatment and genotype is shown. (C) Quantification of mitochondrial fluorescence distribution obtained for mitochondria located 0–1 μm away from the plasma membrane. Results are expressed as mean ± SEM. Data were analyzed using two-way (B) or one way (C) ANOVA and posthoc Bonferroni tests. *p < 0.05, **p < 0.01, ***p < 0.001.