Fig 2: Specific nucleoporins are reduced in G4C2-repeat-expressing adult brain(A) 15-day-old Drosophila adult brain expressing (G4C2)30 repeats (30R) under the control of ELAV-Gene Switch GAL4 (ELAV-GS) induced with 100 mM RU486, stained with Nup214 (green), neuronal marker ELAV (red), and DAPI (blue), (scale bar represents 5 mm, n = 15 brains per genotype). ‘‘*’’ represents non-neuronal cells.(B) Quantification of neuronal Nup214 immunofluorescence is reported as mean ± SEM (one-way ANOVA, followed by Tukey’s test, ****p < 0.0001, n > 60 cells per genotype).(C and D) Nup98 (green), ELAV (red), and DAPI (scale bar represents 2 mm, n = 15 brains per genotype). ‘‘*’’ represents non-neuronal cells. Quantification of neuronal Nup98 fluorescence intensity, data are reported as mean ± SEM (one-way ANOVA, followed by Tukey’s test, ****p < 0.0001, n > 60 cells per genotype).(E) Western blot of adult brains of ELAV-GS/+ and ELAV-GS>30R flies fed RU486.(F) Quantification of western blot in (E) normalized with actin. One-way ANOVA, followed by Sidak’s multiple comparisons (n = 3, ***p < 0.001, ****p < 0.0001, ns = not significant). Error bars indicate ± SEM.

Fig 3: Knockdown of Vps4 restores nuclear nucleoporins and rescues NCT in G4C2-expressing motor neurons(A) Expansion microscopy (ExM) shows Nup98 (green), LaminB (magenta), ELAV (red), and DAPI (blue) of 10-day-old adult brains (scale bar represents 5 mm, n = 10 brain per genotype).(B) Quantification of number of cytoplasmic Nup98 puncta (>0.20 μM area) in (A). Data are reported as mean ± SEM (unpaired t test, ****p < 0.0001, n > 30 cells per genotype).(C) Western blot of cytoplasmic and nuclear fractions from 10-day-old adult brains raised on RU486-containing food.(D and E) Quantification of nucleocytoplasmic ratio from (C). Actin was used to normalize cytoplasmic fraction and LaminB for nuclear fraction (n = 3, Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01).(F) Images showing localization of NLS-NES-GFP (green), LaminB (red), and DAPI in larval motor neurons (scale bar represents 10 mm, n = 10 brains per genotype).(G) Quantification of nucleocytoplasmic (N/C) ratio of NLS-NES-GFP in motor neurons in F (n > 40 cells per genotype; Tukey’s multiple comparisons test, ****p < 0.0001).