Fig. 3

SUN1 and SUN2 alterations in spinal organoid iMNs. Representative images of SUN1 (a, grays) and SUN2 (h, grays) staining in spinal organoids. Islet1 (ISL1, green) expression was used to identify iMNs, while DAPI (blue) labeled the nuclei. b-f. Representative images and line profiles of SUN1 distribution in iMNs from control (b-c) and C9 mutant (d-e) organoids show a disruption in its localization at the NE, which was more frequently observed in iMNs from mutant organoids (quantified in f; Student’s t test, n = 4, **p < 0.01). The yellow dashed lines in a indicate the lines used for the profile plots. g. Quantitative analysis of SUN1 nuclear levels shows a significant reduction in C9 organoids compared to isogenic control (Student’s t test, n = 71 and 31 from 4 independent experiments, *p < 0.05). i-m. Representative images and line profiles of SUN2 distribution in iMNs from control (i-j) and C9 mutant (k-l) organoids show a disruption in its localization at the NE, which was more frequently observed in iMNs from mutant organoids (quantified in m; Student’s t test, n = 4, **p < 0.01). n. Quantitative analysis of SUN2 nuclear levels shows a significant reduction in C9 organoids compared to isogenic control (Student’s t test, n = 71 and 47 from 4 independent experiments, **p < 0.01). o-r. Representative blots and quantification of SUN1 (o-p) and SUN2 (q-r) levels from whole lysates of spinal organoids from C9 and control iPSCs shows a significant reduction in their overall levels (Student’s t test, n = 4 in p, n = 7 and 8 in r, *p < 0.05, ****p < 0.0001). GAPDH was used as loading control. For all, bars are mean and SEM, while violin plots show the distribution of the data with dashed lines indicating median and quartiles. Scale bars: 20 μm in a and h, 10 μm in b, d, i, and k