Fig. 4: TDP-43 pathology disrupts NPC and nuclear lamina morphology and nucleocytoplasmic transport.

a, Immunofluorescence (IF) of endogenous FG-Nups (mAb414, red) and lamin B (magenta) in primary cortical neurons expressing GFP or GFP-tagged TDP-CTF, TDP-43^WT, TDP-43^Q331K, TDP-43^M337V or TDP-43^mtNLS. This experiment was repeated independently five times. Arrows indicate colocalization of TDP-CTF and endogenous FG-Nups; scale bar, 10 μm. b,c, The percentage of transfected cells exhibiting abnormal lamin B staining (invagination, distortion) after transfection with TDP-43 expression vectors (b) or TDP-43 knockdown constructs (c). d, The percentage of transfected cells exhibiting abnormal RanGAP1 staining. e, Super-resolution IF imaging of endogenous Nup98 (red) in N2a cells expressing GFP or GFP-TDP-CTF. Scale bar: 10 μm. f, IF and quantification of mean γH2AX intensity in cortical neurons expressing GFP or GFP-tagged TDP-CTF, TDP-43^WT or TDP-43^Q331K. Calicheamicin (CLM) was added at 5 nM to induce DNA damage. g–i, IF and quantification of N-to-C ratio of a transport reporter encoding NES-tdTomato-NLS in cortical neurons expressing GFP or GFP-tagged TDP-43 constructs (g,h) or with treatment of staurosporine (STS) or importazole (IPZ) Scale bar: 10 μm. i. This experiment was repeated independently three times. j, Quantification of N-to-C ratio of reporter in neurons with TDP-43 knockdown. k, Quantification of the N-to-C ratio of Ran in neurons expressing GFP or GFP-tagged TDP-43 constructs. l,m, IF and quantification of N-to-C ratio of poly(A) RNA. Poly(A) RNA was detected by fluorescence in situ hybridization (FISH) with oligo(dT) probes (red). l, Quantification of newly synthesized protein via metabolic labeling with azidohomoalanine (AHA). Anisomycin (aniso) was added as a translation inhibitor. Scale bar: 10 μm. Graphs represent quartiles (boxes), 50th percentiles (center lines) and range (whiskers). Five independent experiments for b (circles represent each independent experiment; ***P  <  0.001, one-way ANOVA), five independent experiments for c (circles represent each independent experiment; two-sided unpaired t-test), four independent experiments for d (circles represent each independent experiment; *P < 0.05, **P < 0.01, one-way ANOVA), three independent experiments for f (circles represent, for GFP: n = 58, GFP + CLM 5 nM: n = 59, TDP-CTF: n = 61, TDP-43^WT: n = 59 and TDP-43^Q331K: n = 57; ***P < 0.001, one-way ANOVA), three independent experiments for h (circles represent, for GFP: n = 70, TDP-CTF: n = 72, TDP-43^WT: n = 70, TDP-43^Q331K: n = 70, TDP-43^M337V: n = 70, TDP-43^mtNLS: n = 61; **P < 0.01, ***P < 0.001, one-way ANOVA), three independent experiments for i (circles represent, for DMSO: n = 46, IPZ 2.5 μM: n = 45, IPZ 5 μM: n = 45, STS 50 nM: n = 46, STS 250 nM: n = 47; **P < 0.01, ***P < 0.001, one-way ANOVA), four independent experiments for j (circles represent, for nonsilencing control shRNA (shCtrl): n = 77, TDP-43 shRNA (shTDP-43): n = 80; ***P < 0.001, two-sided unpaired t-test), three independent experiments for k (circles represent, for GFP: n = 43, TDP-CTF: n = 44, TDP-43^WT: n = 36, TDP-43^Q331K: n = 51, TDP-43^M337V: n = 46, TDP-43^mtNLS: n = 48; one-way ANOVA), three independent experiments for m (circles represent, for GFP: n = 46, TDP-CTF: n = 61, TDP-43^WT: n = 49, TDP-43^Q331K: n = 51, TDP-43^M337V: n = 60, TDP-43^mtNLS: n = 58; ***P < 0.001, one-way ANOVA), five independent experiments for n (circles represent, for GFP: n = 58, TDP-43^WT: n = 58, TDP-CTF: n = 63, GFP + aniso: n = 59, TDP-43^WT + aniso: n = 55, TDP-CTF + aniso: n = 53; ***P < 0.001, †P < 0.001, two-way ANOVA). Bonferroni’s post hoc test. Full statistical details are provided in Supplementary Table 4. Scale bars in a, e and g: 10 µm.