Fig. 7.

Mechanistic studies of WS disease processes in iPSC-derived OPCs/pmOLs. a RT-qPCR gene expression analysis of ER stress markers BiP, PERK, ATF6, IRE1α and CHOP in WS patient line 1 and 2, and their respective isogenic control OPCs/pmOLs, with/without ER stressors thapsigargin (TH) (2µM, 3 h) or tunicamycin (TM) (1 µg/ml, 24 h). b Protein expression levels of BiP (an indicator of unfolded protein accumulation in the ER lumen) after TM treatment in WS patient OPCs/pmOLs and isogenic controls (% relative to isogenic control). Unpaired t-test; t₄ = 0.7714 for WS patient 1 vs. isogenic 1 and t₄ = 0.6923 for WS patient 2 vs. isogenic 2. c Ratio of spliced to total XBP1 mRNA (an indicator of activation of the unfolded protein response) in WS patient OPCs and isogenic controls after TH treatment (2µM, 3 h). h: hybrid, s: spliced, u: unspliced XBP1 mRNA. Unpaired t-test; t₆ = 1.324 for WS patient 1 vs. isogenic 1 and t₆ = 0.8214 for WS patient 2 vs. isogenic 2. d Bioenergetic profile of mutant and isogenic control OPCs/pmOLs after addition of mitochondrial stressors, oligomycin, FCCP and rotenone. A Seahorse mitostress assay was performed to measure basal respiration, maximal respiration, ATP production, spare respiratory capacity, and extracellular acidification rate (ECAR). e-f Proximity ligation assay assessing proximity between ER (VAPB) and mitochondria (PTPIP51) in WS patient OPCs/pmOLs and isogenic controls, to evaluate MAM integrity. Unpaired t-test; t₃₂ = 1.401 for WS patient 1 vs. isogenic 1 and t₅₆ = 0.9760 for WS patient 2 vs. isogenic 2. Data presented as mean ± SEM. n = 5 for non-treated and TH, and n = 2 for TM (a), n = 3 (b), n = 4 (c), n = 5 for patient line 1 and n = 3–6 for patient line 2 (d), n = 3 with at least 16 images and n > 100 nuclei/condition (e). Scale bar 25 μm (f). Statistics for panel a and d are reported in supplementary table S2. P1: WS patient line 1, I1: isogenic line 1, P2: WS patient line 2, I2: isogenic line 2