Mild TDP-43 overexpression causes accumulation of giant Rab7-positive vesicles. (A) HEK293A WT, HEK-TDP-43-GFP, and HEK-TDP-35-GFP cells were grown in normal conditions before being fixed and immunostained for Rab7. N = 3; scale bar, 10 µm. (B) Quantification of the frequency of giant endosomes in HEK293A WT, HEK-TDP-43-GFP, and HEK-TDP-35-GFP cells. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (C) Quantification of the average size of giant endosomes in HEK293A WT, HEK-TDP-43-GFP, and HEK-TDP-35-GFP cells. ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (D) Distribution of giant Rab7+ve vesicle diameters from WT, HEK-TDP-43-GFP, and HEK-TDP-35-GFP cell lines. N = 3. (E) HEK-TDP-43-GFP (pseudocolored white) cells were grown in normal conditions before being fixed and immunostained for Rab5, LC3B, or LAMP1 (pseudocolored green) and then successively immunostained for Rab7 (pseudocolored red). N = 3; scale bar, 10 µm. (F) HEK-TDP-35-GFP (pseudocolored white) cells were grown in normal conditions before being fixed and immunostained for Rab5, LC3B, or LAMP1 (pseudocolored green) and then successively immunostained for Rab7 (pseudocolored red). N = 3; scale bar, 10 µm in main panels; 2 µm in insets. Error bars in all graphical panels represent standard error of the mean.