Fig. 2
From: LINC complex alterations are a key feature of sporadic and familial ALS/FTD
LINC complex disruption in C9-ALS i3CNs. a. Representative images of SUN1 (grays) in C9-ALS and control i3CNs. DAPI (blue) identified the nuclei, while Phalloidin (green) labeled the actin cytoskeleton. The white boxes indicate the neurons enlarged in the panels on the right. b-d. Plots of line profiles of SUN1 and DAPI intensities normalized to the max intensity in control cells. Mutant cells display frequent mislocalization of SUN1 to either the nucleoplasm or cytoplasm (arrows), quantified in d. The yellow dashed lines in a indicate the lines used for the profile plots (Mann-Whitney t test, n = 4, *p < 0.05). e. The quantification of the nuclear mean fluorescence intensity (MFI) of SUN1 in C9 i3CNs shows a significant reduction in its abundance relative to isogenic controls (Mann-Whitney t test, n = 46 CTRL and C9 neurons respectively from 4 independent differentiations, ****p < 0.0001). f-g. Representative western blot (WB) and quantification of SUN1 levels relative to Histone 3 (H3) expression shows a significative reduction of total SUN1 levels in C9 lines compared to isogenic counterparts (n = 8 independent experiments for both 24a and C52 iPSC isogenic pairs; Student’s t test, **** p < 0.0001). h. Representative images of Nesprin2 (Nesp2, grays) staining pattern in i3CNs from C9 and Ctrl iPSC lines. DAPI (blue) identified the nuclei, while LaminB (LMNB, green) labeled the nuclear lamina. The white boxes indicate the neurons enlarged in the panels on the right. i-k. Plots of line profiles of Nesprin2 and DAPI intensities normalized to the max intensity in control cells. Mutant cells display frequent mislocalization of Nesprin2 to either the nucleoplasm or cytoplasm (arrows), quantified in k. The yellow dashed lines in h indicate the lines used for the profile plots (Student’s t test, n = 4, *p < 0.05). l. The quantification of the Nesprin2 relative nuclear MFI shows a significant reduction in its abundance in C9 i3CNs compared to isogenic controls (Mann-Whitney t test, n = 31 and 38 for CTRL and C9 neurons from 4 independent differentiations, * p < 0.05). Scale bars: 20 μm in the main panels, 10 μm in zoomed-in images. For all, bars are mean and SEM, while violin plots show the distribution of the data with dashed lines indicating median and quartiles