Fig. 6

TDP-43 functions in classical NHEJ, whereas ALS-associated mutants lose this DNA repair function. a Schematic illustration of total NHEJ reporter, EJ5GFP (left panel), and alternative NHEJ, EJ2GFP (right panel) [38]. Successful repair of I-SceI digestion leads to restoration of GFP gene expression for each reporter. b Cells were co-transfected with digested NHEJ reporter EJ5GFP and either mCherry only (mCherry), or TDP-43 wildtype, or mutants A315T or Q331K, via nucleofection. The efficiency of total NHEJ (classical and alternative) was determined by the % of cells displaying GFP fluorescence. Quantification of GFP-positive cells in EJ5GFP transfectants indicated more total NHEJ in cells expressing wildtype TDP-43 compared to those expressing mutants or controls. One-way ANOVA, Mean ± SEM, *p < 0.05, ***p < 0.001. Scale bar 10 μm. At least 100 cells per group were counted, N = 6. (c) Cells were co-transfected with digested NHEJ reporter EJ2GFP and either mCherry only (mCherry), or TDP-43 wildtype, or mutants A315T or Q331K, via nucleofection. The efficiency of alternative NHEJ was determined by the % of cells displaying GFP fluorescence. Quantification of the number of GFP-positive cells in EJ2GFP transfectants indicates no significant differences in alternative NHEJ between cells expressing TDP-43 wildtype, mutants or controls, indicating that TDP-43 does not function in alternative NHEJ. One-way ANOVA, Mean ± SEM, p > 0.05. At least 57 cells per group were counted, N = 3