Figure 1.

Nuclear RFP-TDP-43^wt and cytoplasmic RFP-TDP-43^208–414 expression in cultured cortical rat astrocytes. (A–C) Representative fluorescence images of astrocytes immunostained with antibodies against endogenous TDP-43 (green) and labelled with DAPI (blue) in (A) control (non-transfected), (B) RFP-tagged wild-type TDP-43-expressing astrocytes (RFP-TDP-43^wt; red) and (C) RFP-tagged C-terminal fragment of TDP-43-expressing astrocytes (RFP-TDP-43^208–414; red). Note the red fluorescent inclusions in the cytoplasm of astrocytes expressing RFP-TDP-43^208–414 and nuclear expression of RFP-TDP-43^wt. Scale bar, 20 µm. (D) Colocalization (%) between RFP fluorescence signal (red) and Alexa Fluor⁴⁸⁸-labelled TDP-43 antibody (green) in astrocytes transfected with RFP-TDP-43^wt or RFP-TDP-43^208–414. (E) Colocalization (%) between the total cellular TDP-43 antibody signal and the nuclear DAPI stain in control cells and in cells transfected with RFP-TDP-43^wt or RFP-TDP-43^208–414. Note the low percentage of TDP-43 antibody colocalization with the nuclear DAPI stain in RFP-TDP-43^208–414-expressing astrocytes, indicating that in these cells, most of the TDP-43 protein resides in the cytoplasm. (F) The percentage (%) of DAPI and TDP-43-colabeled nuclei in non-transfected astrocytes (no visible RFP signal) in control experimental group (Control; n = 99 cells from 28 images) and in non-transfected astrocytes around the astrocytes expressing the RFP-constructs in RFP-TDP-43^wt (n = 62 cells from 18 images), and RFP-TDP-43^208–414 (n = 77 cells from 19 images) experimental groups. The numbers by the error bars indicate the number of cells (D, E) or number of cell nuclei (F) analysed. Data are presented as means ± SEM and acquired from at least two different animals. Each experiment (i.e. coverslip) was performed in duplicate, multiple cells were recorded per coverslip. ***P ≤ 0.001, Mann-Whitney U test (D); ***P ≤ 0.001, Kruskal-Wallis one-way ANOVA on ranks, followed by Dunn’s test (E).