Fig. 4.

Expanded G4C2r expression impairs autophagic flux by inhibiting autophagosome–lysosome fusion in flies. A Representative western blots and quantification of fly brain lysates of indicated genotypes showing that the cleaved product band (free GFP cleaved from GFP-mCherry-atg8) is largely missing from motor neurons expressing (G4C2)₃₀ driven by OK371-GAL4 (i, ii), suggesting an inhibition of autophagy activity in these neurons (numbers above bars, numbers of flies; statistical analysis, two-tailed unpaired t test). Note: each lane is loaded with the lysate from a single fly brain, and the cleaved product may be too low to detect in samples expressing (G4C2)₃₀. B, C Representative western blots of the ref(2)P protein (homologue of human SQSTM1/p62) showing an increased level in larvae expressing (G4C2)₃₀ (B) and pupae (C) (numbers in bars, numbers of flies; statistical analysis, two-tailed unpaired t test). D Representative immunofluorescence images of GFP-mCherry-atg8 puncta and quantification of the autophagosome/autolysosome ratio (red + green + puncta : red + green – puncta) in fly larvae (arrows, putative autolysosomes; arrowheads, putative autophagosomes; n, number of individual cells in >20 flies per group; statistical analysis, two-tailed unpaired t test). In all plots, bars indicate the mean ± SEM; n.s. P > 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).