Fig. 3: Mimicking the hypo-phosphorylation of TDP-43 C-terminus antagonizes its nuclear retention in HeLa cells.
From: TDP-43 nuclear retention is antagonized by hypo-phosphorylation of its C-terminus in the cytoplasm
a Left panel: Subcellular distribution of indicated HA-tagged phosphomimetic mutants expressed in HeLa cells 24 h after transfection. Poly(A) mRNA in green; anti-HA in red. Middle panel: Automatic measurement of the ratio of the HA-tagged TDP-43 cytoplasmic to nuclear level under indicated conditions. Right panel: Same measurement as the middle panel with a selection of cells displaying similar nuclear TDP-43 levels. Each dot represents the mean value of a single well (96-well plate). Error bars indicate SEM and the asterisks indicate statistical significance with ****p < 0.0001, ns. non-significant, as measured by ANOVA test from n = 6 wells. Arrows indicate representative cells. Scale bar: 20 μm. b Same as (a) 48 h post-transfection in HeLa cells treated with Actinomycin D (ActD) or not to promote TDP-43 translocation into the cytoplasm. Error bars indicate SEM and the asterisks indicate statistical significance with **p < 0.01, ns. non-significant, as measured by ANOVA test from n = 8 wells. Arrows indicate representative cells. Scale bar: 20 μm. c Analysis of the skipping of CFTR exon 9, which is controlled by TDP-43, using the indicated minigene reporter in stable shRNA HEK cells that no longer expressed endogenous TDP-43 and after adding back the expression of HA-tagged TDP-43 mutants. Left panel: Representative image of stable shRNA HEK cells under indicated conditions. Upper right panel: Splicing reporter used to detect the skipping of CFTR exon 9. Lower right panel: Quantitative analysis of the splicing efficiency and anti-HA fluorescence intensity under the indicated condition at the single cell level. Each dot represents the average values in selected HEK cells in a single well. Quantitative analysis was performed without any selection (left) or with cells expressing the same level of HA-tagged protein in the nucleus as shown in the panel (right). Error bars indicate SEM and the asterisks indicate statistical significance with *p < 0.05 as measured by unpaired Student’s t-test and ****p < 0.0001, ns non-significant, as measured by ANOVA test from n = 8 wells. Scale bar: 50 μm.
