Figure 8.

Targeting of TDP-43 to aggresomes was impaired in C9orf72 fibroblasts. (A) Human patient-derived fibroblasts were treated for 24 h with 20 μM MG132 or vehicle control (DMSO) and then immunostained with phospho-TDP-43 (pTDP-43, red) and p62 (green) antibodies. Examples of pTDP-43-positive/p62-positive aggresomes were indicated by white arrows. Examples of areas where pTDP-43 had some co-localization with p62 were indicated by white arrow heads. Nuclei were visualized by DAPI stain (blue). (B, C) The intensity of pTDP-43 (B) and p62 (C) immunostaining in patient fibroblasts was normalized to DAPI intensity and quantified as described in Materials and Methods section. (D) The percentages of pTDP-43 co-localized with p62 in patient fibroblasts were quantified as described in the Materials and Methods section. (E) Human patient-derived fibroblasts were treated for 24 h with 20 μM MG132 or vehicle control (DMSO) and then immunostained with C-terminal TDP-43 (TDP-43, red) and p62 (green) antibodies. Examples of TDP-43-positive aggresomes were indicated by white arrows. Examples of areas where TDP-43 had some co-localization with p62 were indicated by white arrow heads. Nuclei were visualized by DAPI stain (blue). The percentages of TDP-43 co-localized with nucleus as visualized by DAPI stain (F), TDP-43 co-localized with p62 (G) and p62 co-localized with TDP-43 (H) were quantified as described in the Materials and Methods section. All statistical analyses in this figure were carried out using each case as the experimental unit as described in the Materials and Methods section. The mean for each experimental group was represented by a horizontal line. ^aP < 0.05 when compared with the corresponding vehicle-treated fibroblasts, ^bP < 0.05 when compared with the corresponding control fibroblasts, ^cP < 0.05 when compared with the corresponding sporadic disease fibroblasts based on two-way ANOVA and post-hoc Tukey’s test. C9, C9orf72; CTRL, control; pTDP-43, phosphorylated TDP-43.