Fig. 2

The N-terminal half of KPNB1 is required and sufficient to reduce TDP-CTF aggregation and toxicity. a (Top) Schematic domain structure of the N-terminal half (HEAT repeats 1–9, H1–9) and C-terminal half (HEAT repeats 10–19, H10–19) of KPNB1. (Bottom) Immunofluorescence (IF) of SH-SY5Y cells co-expressing mCherry or mCherry-TDP-CTF with GFP, GFP-KPNB1 full-length (FL), H1–9 or H10–19. Both full-length and the N-terminal half of KPNB1 reduce TDP-CTF aggregation, whereas the C-terminal half of KPNB1 co-aggregates with TDP-CTF in the cytoplasm. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. b Western blot analysis and quantification of insoluble mCherry-TDP-CTF protein levels in SH-SY5Y cells expressing GFP, GFP-KPNB1 full-length (FL), H1–9 or H10–19. Both full-length and the N-terminal half of KPNB1 significantly reduced insoluble TDP-CTF levels. β-tubulin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (**p < 0.01, ***p < 0.001, n = 4). c Quantification of TDP-CTF-induced cytotoxicity upon overexpression of KPNB1. mCherry or mCherry-TDP-CTF were co-expressed in SH-SY5Y cells with GFP, GFP-KPNB1 full-length (FL), H1–9 or H10–19. Forty-eight hours after transfection, cells were labelled with the Live-or-Dye™ 640/662 dye and dying cells were quantified for each group. Both full-length and the N-terminal half of KPNB1 significantly ameliorated TDP-CTF-mediated toxicity. Statistical analysis was performed with two-way ANOVA and Bonferroni’s post hoc test (four independent experiments; ***p < 0.001. mCherry: GFP (n = 400), GFP-KPNB1^FL (n = 405), GFP-KPNB1^H1–9 (n = 426), GFP-KPNB1^H10–19 (n = 406); mCherry-TDP-CTF: GFP (n = 405), GFP-KPNB1^FL (n = 401), GFP-KPNB1^H1–9 (n = 405), GFP-KPNB1^H10–19 (n = 400)). d IF of SH-SY5Y cells co-expressing mCherry or mCherry-TDP-CTF with GFP-KPNB1 H1–9 or C-terminal deletion constructs. TDP-CTF appeared nucleocytoplasmic with KPNB1 H1–9 and H1–8, while it formed small aggregates in presence of KPNB1 H1–7 or H1–6. Arrowheads indicate co-aggregation. Hoechst staining was used to outline nuclei. Scale bar: 5 μm. e, Western blot analysis and quantification of insoluble mCherry-TDP-CTF protein levels in SH-SY5Y cells expressing GFP, GFP-KPNB1 full-length (FL), H1–9 or C-terminal deletion constructs. KPNB1 H1–8 was the most active fragment in reducing insoluble TDP-CTF levels. β-actin was used as a loading control. Statistical analysis was performed using one-way ANOVA and Bonferroni’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001, n = 4)