Fig. 10
VCP inhibition or VCP disease mutations enhance TDP-43 seeding in cells. A Representative FRET confocal microscopy images of TDP-43 biosensor line (TDP-43 Clover/Ruby) treated with empty Lipofectamine (left, ctrl), 50nM TDP-43 monomer (middle) and 50nM TDP-43 PFF (right) after 48 hours. Scale bar= 10μm. B Tracing of FRET signal via flow cytometry. FRET+ gate (Clover vs FRET) was drawn from empty Lipofectamine treated cells with no aggregation. C Quantitation of integrated FRET signal is measured by % FRET+ cell * Median Fluorescent Intensity (FRET+ cells). **** p < 0.0001, n.s., no significance by one-way ANOVA. error bars are ±S.E.M. D Graph of FRET efficiency from TDP-43 biosensors were treated with TDP-43 PFF at different concentration and harvested after 48 hours (each dot represents triplicates in each condition). E Immunoblot for TDP-43 CTF (anti-GFP) from detergent soluble and insoluble lysates of TDP-43biosensor cells treated with TDP-43 monomer or PFF and then harvested after 48 hours. Note that the RIPA insoluble fraction accumulated high molecular weight TDP-43 positive multimers. GAPDH is a loading control. F Normalized FRET from TDP-43 biosensors co-treated with TDP-43 monomer or PFF and NMS-873 (5μm) or DMSO vehicle control for four hours followed by washout. Cells were harvested at 48 hours after the treatment, and analyzed the same as 10C. FRET signal is normalized to DMSO 100nM PFF treated group. (n >4 repeats for each group. ****p < 0.0001; n.s. no significance, two-way ANOVA with Šidák correction) G TDP-43 biosensors were transfected with plasmids expressing VCP-WT, or one of two disease mutations (R155H and A232E) for 24 hours and then treated with TDP-43 PFF (10nM) for 48 hours. FRET efficiency is all normalized by VCPWT. (n=9 repeats for each group. *p <0.05, **p < 0.01; ****p < 0.0001; one-way ANOVA with Dunnett’s correction)