Figure 5.

A complete PQC system blockage boosted the extracellular secretion of C-terminal TDP-43 fragments. (A,B) NTA distribution of LVs (A) and SVs (B) isolated from NSC-34 cells treated o/n with both MG132 (10 μM) and NH₄Cl (20 mM) (DT = double-treated samples). Graphs represent the mean of n = 3 biological replicates; x-axis = vesicles dimension, expressed in nm; y-axis = vesicles concentration, expressed as ratio between the number of vesicles and the number of secreting cells. Data show that PQC blockage statistically increased the secretion of 190–230 and 270–310 nm large vesicles (A) (* p < 0.05, Welch’s t-test). (C–F) WB analysis (C) and relative quantifications (D–F) of total RIPA-extracted proteins from NSC-34 cells, LVs and SVs, untreated or treated o/n with MG132 (10 μM) and NH₄Cl (20 mM). Samples were analyzed for TDP-43 species, MAP1LC3B-II, BAG3, HSPB8, BAG1, ALIX, INT β1 and HIS H3. Bar graphs represent the mean optical density ± SD of a specific protein normalized on the optical density of the internal housekeeping protein (HIS H3 for cells and SVs; INT β1 for LVs) and reported in comparison to the corresponding untreated sample (ctrl), for three biological replicates. (* p < 0.05, ** p < 0.01, unpaired one-tailed t-test).