F: A HEK293 cell line expressing the proteostasis reporter UbG76V-GFP was transiently transfected with tagRFP, tagRFP-TDP-25 wild-type, tagRFP-TDP-25mutant, tagRFP-TDP-43 wild-type or tagRFP-TDP-43 mutant. UbG76V-GFP levels were analyzed by flow cytometry 72 h after transfection. The relationship of binned tagRFP fluorescence and UbG76V-GFP fluorescence is plotted. Values shown are means ± SD obtained from n=4 independent experiments. For clarity, adjusted p-values of pairwise comparisons within each tagRFP intensity bin are shown in Fig. EV3C. Note that at lower expression levels (bin < 30) both TDP-25 variants induce higher UbG76V-GFP levels than their respective full-length counterparts indicative of stronger impairment of proteostasis. The full-length variants show meaningful accumulation of the reporter levels only at the high-expression levels. The mutant versions of TDP-25 as well as TDP-43 lead to an increased reporter signal at higher expression levels compared to wild-type variants. 