Journal: eLife

Article Title: Rapid and inducible mislocalization of endogenous TDP43 in a novel human model of amyotrophic lateral sclerosis

doi: 10.7554/eLife.95062

Figure Lengend Snippet: ( A ) Schematic depicting the knock-in of the nanobody (control or nuclear export signal , NES) into the human AAVS1 safe harbour locus of our E8 homozygous TDP43-GFP cell line. Nanobody expression was under the control of a doxycycline (Dox)-inducible promoter. Addition of Dox is expected to induce TDP43 mislocalization in the TDP43-GFP-NES motor neurons (MNs) but not in the TDP43-GFP-Control (TDP43-GFP-CTRL) MNs. Panel created in BioRender . ( B ) Immunofluorescent staining showing TDP43 localization in our TDP43-GFP-CTRL or TDP43-GFP-NES line in response to Dox. Mislocalization only occurs in the TDP43-GFP-NES line with Dox treatment, while all other conditions maintain nuclear TDP43. MNs were fixed and stained at day 35, 15 days post-Dox addition. Scale bar = 50 µm. ( C ) TDP43 localization in the TDP43-GFP-NES or –CTRL cell lines at day 40, 20 days post-Dox addition. The box highlights cytoplasmic TDP43 puncta in the NES line. Scale bar = 20 µm. ( D ) Western blots of total and phosphorylated TDP43 in TDP43-GFP-NES or –CTRL cell lines at day 40, 20 days post-Dox addition. Alpha-tubulin was used as a loading control. ( E ) Quantification of total and phosphorylated TDP43-GFP from Figure D. Total TDP43-GFP remains stable, while there is a significant increase in phosphorylated TDP43-GFP in the NES lines. Each sample was normalized to alpha-tubulin, pTDP43 samples were also normalized to total TDP43 levels. Replicates are three independent differentiations of TDP43-GFP-NES or –CTRL. pTDP43-GFP=phosphorylated TDP43-GFP. * indicates p<0.05. Error bars indicate SEM. Figure 3—source data 1. PDF of labelled uncropped western blots shown in . Figure 3—source data 2. unedited original files for western blots shown in .

Article Snippet: Homozygous TDP43-GFP iPSCs derived from clone E8 were transfected with the donor vectors, and a sgRNA targeting the human AAVS1 locus (Addgene 126582, a kind gift from Michael Guertin ).

Techniques: Knock-In, Control, Expressing, Staining, Western Blot