Fig. 6: Selected kinase inhibitors antagonize the nuclear retention of TDP-43 in contrast with phosphatase inhibitors.
From: TDP-43 nuclear retention is antagonized by hypo-phosphorylation of its C-terminus in the cytoplasm
a Left panel: Subcellular distribution of HA-tagged wild type TDP-43 in HeLa cells after treatment with the indicated compounds during 4 h (10 µM). Poly(A) mRNA in green (poly-dT probe) and anti-HA in red. Arrows indicate representative cells. Scale bar: 50 μm. Right panel: Automatic measurement of the cytoplasmic:nuclear ratio of HA-tagged TDP-43 under indicated conditions. Each data point represents the mean ratio value obtained from a single well in a 96-well plate. As control, the cytoplasmic:nuclear ratio of the hyper-phosphomimetic mutant was measured under the same condition. Error bars indicate SEM and the asterisks indicate statistical significance with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns non-significant as measured by ANOVA test from n = 4 wells. b Left panel: Subcellular distribution of HA-tagged wild type TDP-43 in undifferentiated mouse motor neuron-like hybrid cell line, NCS-34 cells, after treatment with the indicated compounds for 4 h (3 µM). Poly(A) mRNA in green; anti-HA in red. Scale bar: 50 μm. Right panel: Automatic quantification of the nuclear:cytoplasmic ratio of HA-tagged TDP-43 under indicated conditions. Each data point represents the mean ratio value obtained from 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. c Left panel: Recruitment of HA-tagged G146A mutant in cytoplasmic condensates in HeLa cells treated with the indicated compounds during 4 h (10 µM). Anti-HA in red. mRNA in green. Scale bar: 50 μm. Zoom scale bar: 10 μm. Right panel: Automatic measurement of the enrichment of HA-tagged G146A or mRNA in cytoplasmic condensates versus cytosol under indicated conditions. Error bars indicate SEM and the asterisks indicate statistical significance with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns non-significant as measured by ANOVA test from n = 4 wells.
