Figure 1. ER stress and proteasome inhibition cause redistribution of wildtype and ALS-linked mutant TDP-43.

(A) Neuro2a cells were transfected with the construct indicated to the left of each series of panels (mCherry alone, wildtype TDP-43-mCherry, or mutant D169G, G294A, A315T, Q331K, M337V or N390D TDP-43-mCherry), and fixed at 48 h post-transfection. mCherry fluorescence is shown in the left panels, and Hoechst nuclei stain is shown in the merged right panels. Note the largely nuclear distribution of wildtype and mutant TDP-43-mCherry, but with low levels of non-nuclear mCherry fluorescence in some cells, indicated by arrows. (B) Immunoblot of cell lysates expressing mCherry alone or wildtype or mutant TDP-43-mCherry showing equal expression levels of all proteins, and levels of endogenous (End.) TDP-43. Approximate molecular weight markers are shown on the right. (C) Quantification of the effect of thapsigargin or MG132 treatment on the percentage of Neuro2a cells with cytoplasmic mCherry fluorescence. Results are expressed as mean ± SEM, n = 3, *p<0.05 versus respective vehicle treated controls by two-way ANOVA with Bonferroni's post-test. (D) Neuro2a cells were transfected as in A and treated with 100 nM thapsigargin for 24 h prior to fixation. (E) Neuro2a cells were transfected as in A and treated with 10 µM MG132 for 24 h prior to fixation. Note the increased non-nuclear distribution of wildtype and mutant TDP-43-mCherry with both thapsigargin and MG132 treatment, indicated by arrows. All scale bars represent 10 µm.