Fig. 11
VCP disease mutations enhance TDP-43 seeding in neurons. A Immunofluorescent staining for pTDP-43 (red), Tuj1 (green), and nuclei (blue) in primary hippocampal neurons treated with 10nM TDP-43 monomer or 10nM TDP-43 PFF for 5 days. Scare bar=10μm. B Immunoblot for TDP-43 from detergent soluble and insoluble lysates of HNs treated with 10nM TDP-43 monomer or 10 nM PFF and then harvested after 1 or 5 days. Note that the RIPA insoluble fraction has high molecular weight TDP-43 positive multimers. pan 14-3-3 is a loading control. C Quantitation of area of pTDP-43 immunofluorescence in HNs after 4 hours or 5 days after 10nM TDP-43 PFF treatment. D Quantitation of area of pTDP-43 immunofluorescence treated with the indicated concentrations of TDP-43 PFF and harvested at 5 days. E Immunofluorescent images for pTDP-43, SQSTM1, and TIA-1 from neurons treated with TDP-43 monomer or TDP-43 PFF for five days. Scare bar=10 μm. F Immunofluorescence for phospho-TDP-43 (red) and Tuj1 (green) in HNs from wild-type mice or mice carrying a VCP-R155H knockin allele (VCPR155H/WT) treated with TDP-43 for PFF for 5 days. (Scare bar= 20μm). G Quantitation of phospho-TDP/Tuj1 staining (Neurons coming from 3 and 4 independent cultures from WT and VCPR155H/WT embryos. Outlier is removed by ROUT method, Q=1%, followed by Student’s t test. n=4, WT and VCPR155H/WT group respectively. *p<0.05.)