Fig. 2: Leak through the RyR receptor is responsible for ER Ca²⁺ loss in WFS1-deficient neurons.

a Thapsigargin-induced Ca²⁺ release from the ER to cytoplasm is lower in WFS1- and CISD2-deficient neurons. The left panel depicts the typical Ca²⁺ transients obtained after treatment with 2 µM Thapsigargin in Ca²⁺-free media in the presence of 0.5 mM EGTA and the effect of subsequent reintroduction of external 10 mM Ca²⁺. The right panel shows the peak values of these transients before and after Thapsigargin or Ca²⁺ treatments. n = 14 (scrambled and Wfs1 shRNA) or 13 (Cisd2 shRNA) neurons, Brown-Forsythe ANOVA and Dunnett’s T3 multiple comparisons test. b, c RyR2 knock-down restored the ER and axoplasmic Ca²⁺ levels in the axons of the WFS1-deficient neurons. n = 120 (b), n = 70, 58, 58 or 59 (c) neurons, one-way ANOVA, and Šídák’s multiple comparisons test. d, e Treatment with 20 µM Azumolene (48 h) restored the basal ER and axoplasmic Ca²⁺ levels in the axons of WFS1-deficient neurons. n = 30, 29, 30 or 30 (d), n = 29, 30, 30 or 30 (e) neurons, One-way ANOVA and Šídák’s multiple comparisons test. f, g Treatment with 5 μM Rycal S107 (48 h) restored the basal ER and axoplasmic Ca²⁺ levels in WFS1-deficient neurons. n = 30 (f), n = 88, 87, 90 or 89 (g) neurons, one-way ANOVA and Šídák’s multiple comparisons test. h, i IP₃R1 and IP₃R3 knock-down did not restore the basal ER and axoplasmic Ca²⁺ levels in WFS1-deficient neurons. n = 90 (h), n = 60, 57, 118 or 60 (i) neurons, Brown-Forsythe ANOVA and Dunnett’s T3 multiple comparisons test or Kruskal–Wallis test & Dunn’s multiple comparisons test. Data are presented as mean ± SEM.