Fig. 7: Kinase inhibitors and the hypo-phosphomimetic mutations (18A) impaired TDP-43 nuclear retention because of cytoplasmic TDP-43 self-assembly.
From: TDP-43 nuclear retention is antagonized by hypo-phosphorylation of its C-terminus in the cytoplasm
a Scatter plot of subcellular distribution of HA-tagged wild type and hypo-phosphomimetic TDP-43. A linear regression with a high R² (0.675) shows that the level of cytoplasmic and nuclear hypo-phosphomimetic mutant increase linearly, as expected for passive diffusion. On the other hand, in HeLa cells expressing wild-type TDP-43, we observed a fraction of the cells with increased TDP-43 nuclear levels. Each dot represents a single cell. The outlines were done by hand to help the reader. b Left panel: Representative images of HeLa cells treated with the indicated compounds during 4 h (10 µM). Red, endogenous TDP-43. Green, anti-phospho TDP-43 (Ser 409/410). Arrows indicate an increased brightness of anti-phospho-TDP-43 positive nuclear granules. Arrows show representative nuclear granules. Scale bar: 20 µm. Right panel: Quantification of fluorescence ratio of anti-phospho TDP-43 inside versus outside nuclear granules. Only nuclei were selected for this analysis. Error bars indicate SEM and the asterisks indicate statistical significance with *p < 0.05, **p < 0.01, ****p < 0.0001, ns. non-significant as measured by ANOVA test from n = 4 wells. c Same as (b) with mouse motor neuron-like hybrid cell line NSC-34. Left panel: Representative images of NSC-34 cells. Red, tubulin. Green, anti-phospho TDP-43. Arrows show representative nuclear granules. Scale bar: 30 µm. Right panel: statistical analysis. The effect was assessed for four concentrations of compounds. Error bars indicate SEM and the asterisks indicate statistical significance with **p < 0.01, ***p < 0.001, ****p < 0.0001, ns non-significant as measured by ANOVA test from n = 4 wells. d Left panel: Representative images of HeLa cells expressing HA-tagged TDP-43 or indicated mutant. Arrows indicate representative cytoplasmic TDP-43-rich granules. Scale bar: 30 μm. Zoom scale bar: 20 μm. Right panel: Quantification of the anti-HA intensity in detected cytoplasmic spots and the density of spots per cytoplasm area. Each dot represents the mean value obtained over the selected cells in a single well. Error bars indicate SEM and the asterisks indicate statistical significance with ***p < 0.001, ****p < 0.0001, ns non-significant as measured by ANOVA test from n = 8 wells.
