VPS4A ^E228Q increases TDP-43 stability and promotes cytoplasmic mislocalization. (A) HEK293A WT cells were transfected with EGFP-VPS4A^E228Q plasmid (pRB475) or mock transfected and grown in normal conditions for 48 h. Cells were then treated with 0.3 mg/ml cycloheximide for the indicated amounts of time, and TDP-43 levels were detected by western blot with anti–TDP-43 antibodies and normalized to the level of the GAPDH loading control. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (B) HEK293A WT cells were either mock transfected (top row) or transfected with EGFP-VPS4A^E228Q plasmid (next two rows, to show phenotypic diversity) and grown in normal conditions for 48 h before being fixed and immunostained with anti–TDP-43 antibodies. White arrowheads indicate TDP-43 and EGFP-VPS4A^E288Q colocalization; red arrowhead indicates example of TDP-43-distinct foci; green arrowheads indicate examples of distinct EGFP-VPS4A^E288Q foci/vesicular structures. N = 3; scale bar, 10 µm. (C) Quantification of mean nuclear and cytoplasmic TDP-43 signal intensities in HEK293A WT cells and cells transfected with EGFP-VPS4A^E228Q plasmid. *, P < 0.05; ***, P < 0.001 by Mann–Whitney U test (nuclear) or Welch’s two-tailed T test (cytoplasmic); N = 30. (D) Quantification of the percentage of cells with cytoplasmic TDP-43 foci in HEK293A WT cells and cells transfected with EGFP-VPS4A^E228Q plasmid. **, P < 0.01 by Welch’s two-tailed T test; N = 3. (E) HEK293A, HEK-TDP-43-GFP, and HEK-TDP-35-GFP cells were seeded at equal cell numbers and transfected with VPS4A^E228Q plasmid or mock transfected, and cell proliferation was compared after 48 h ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. Error bars in all graphical panels represent standard error of the mean. Source data are available for this figure: SourceData F4.