Fig. 5: Identification of kinase/phosphatase inhibitors affecting TDP-43 C-terminus self-assembly in the cytoplasm.
From: TDP-43 nuclear retention is antagonized by hypo-phosphorylation of its C-terminus in the cytoplasm
a Left panel: Mixing score between wild-type TDP-43 and 18E mutant and between 18E and 18A mutants in a 96-well plate. The measured Z’ factor is 0.57, which corresponds to a very good assay for compounds decreasing the mixing score. Right panel: Representative image of the mixing of indicated proteins along the microtubule networks in HeLa cells. Scale bar: 50 μm. b Summary of the results of the primary screen. c Scatter plot representing the mixing score for all the tested compounds. Red, kinase inhibitors, Blue: Phosphatase inhibitors, Gray: hits. HeLa cells were treated for 4 h with 10 µM of the indicated compounds. d Scatter plot of the mixing score for treatments with DMSO (control), kinase, and phosphatase inhibitors. Globally, kinase inhibitors decrease while phosphatase inhibitors increase the mixing of the hyper-phosphomimetic mutants (18E) with wild-type TDP-43. p values are indicated. ANOVA test. e Representative image of the mixing of wtTDP-43 with 18E after indicated treatments. Scale bar: 50 μm. f Mixing score changes when comparing the mixing of wild-type TDP-43 with either 18E or 18A mutant under indicated treatment. The mixing scores were normalized to 1 under control conditions (DMSO) to help detecting a putative differential mixing. Changes in the normalized mixing score are represented for 13 kinase and 2 phosphatase inhibitors selected from the primary screen. Statistical significance of the bidirectionality was probed with an ANOVA test. Error bars indicate SEM and the asterisks indicate statistical significance with *p < 0.05, **p < 0.01, ***p < 0.001, ns non-significant, as measured by ANOVA test from n = 4 wells. For each condition, the mean mixing score was measured in 4 wells (96-well plates). Kinase inhibitors (in red). Phosphatase inhibitor (in blue). g IC50 curves of the mixing score obtained from the analysis of 4 wells per condition. Incubation time: 4 h. The IC50 values are reported for 4 compounds.
