Fig. 3
Dysregulation of cytosolic calcium homeostasis in OPA1 R445H iPSC-RGCs in response to ER calcium release. (a) Representative live cell confocal images of cytosolic calcium levels in isogenic control and R445H neurons at D42 stained with Fluo4 at baseline, and after treatment with 1.5 µM thapsigargin and 1.2 mM CaCl2. Scale bar, 20 μm. (b & c) Quantification of fold-change in Fluo4 fluorescence intensity over time normalised to baseline values, denoted F0. 1.5 µM thapsigargin and 1.2 mM CaCl2 were added at the indicated time points. Neurons were maintained in calcium-free recording buffer until addition of CaCl2. Grey lines show Fluo4 fluorescence intensity in individual cells from a representative experiment, blue/red lines the mean average normalised Fluo4 fluorescence intensity. (d-i) Quantification of initial slope, amplitude and decay slope of Fluo4 fluorescence intensity in response to 1.5 µM thapsigargin and 1.2 mM CaCl2 in isogenic control R445H and iPSC-RGCs. Isogenic control, n = 140; R445H, n = 190 cells sampled from three independent differentiations. * = p < 0.05; **** = p < 0.0001, Mann-Whitney tests. Box plots show median (middle line), 25th-75th percentile (box) and min/max values (whiskers)