Fig 1: Impairment of nucleo-cytoplasmatic FUS shuttling leads to DNA damage, neurodegeneration and aggregate formation. a Time course of nuclear FUS withdrawal after addition of PARP1 inhib. (top gallery) or etoposide (eto, bottom gallery) in WT FUS-GFP MNs at 21 DIV. b Distinct inhibitory kinetics of FUS-dependent DNA repair through etoposide and PARP1 inhib: recruitment-withdrawal of FUS-GFP to nuclear laser cuts as for Fig. 6a on WT FUS-GFP MNs after PARP1 inhibitor (top) or etoposide (bottom) addition at indicated time points, i.e. before and after loss of nuclear FUS (corresponding red boxes in a), bars: 10 µm (Supplementary Movie 12). c Quantification of b. d FUS-GFP recruited to Laser cut in MNs double-treated 24 h with compounds as indicated (Supplementary Movie 13). Note the inhibition of recruitment to the cut (boxed areas) in mutant FUS-GFP P525L cells (far left) through double treatment with DNA-PKi and PARP1i whereas DNA-PKi alone led to a rescue (Fig. 6c, Supplementary Movie 6), suggesting that PARP1 functions upstream of DNA-PK in DNA damage response. Furthermore, note that inhibiton of recruitment through etoposide or arsenite in WT FUS-GFP (Fig. 6c, Supplementary Movie 6) was reverted through double treatment with either DNA-PKi or AdOx, suggesting a potent counterbalancing of etoposide/arsenite-driven displacement of FUS from the nucleus, thereby rescuing FUS recruitment to the cut (boxed areas). e Quantification of d. f Compounds impacted on nuclear FUS-GFP levels in MNs: double treatments Mut versus WT as for d. Note how DNA-PK inhib. or AdOx drove FUS-GFP back into the nucleus under conditions that normally lead to nuclear export of FUS (i.e. PARP1, etoposide or arsenite treatment, Fig. 6e). g Western blot (WB) analysis of markers for c-NHEJ DNA damage response (phospho and total DNA-PK, KU70 & KU80) and a-NHEJ (LIG1, PARP1 total and cleaved) on total lysates of MNs treated for 72 h as indicated. GFP, β-actin served as loading controls. h Densiometric quantification of g, WT (blue) versus FUS (red), mock only. Graphs show fold change over mean WT (mock baseline = 1) to reveal phenotypic differences of quasi untreated FUS over WT. i Densiometric quantification of g for all treatments. Graphs show fold change over respective cell line mock to reveal the compound response of each line (WT, blue, versus FUS, red) over its respective mock baseline ( = 1). Statistics (h, i): data are plotted as mean, error bars = STDEV, one-way ANOVA with post-hoc Bonferroni test (*, **, ***P values of 0.05, 0.01, and 0.001, respectively, N = 4)