Fig. 3

Progranulin-null lysosomes from human neurons and mouse brains contain increased hydrolases levels but have decreased enzymatic activity. A Schematic of intact lysosomal isolation (Lyso-IP) proteomics in GRN KO vs. WT i³Neurons. B Volcano plot of Lyso-IP proteomics showing protein changes related to protein catabolic processes (red), lysosomal pH (blue), and hydrolase activities (green). Nonspecific labeling proteins were removed from the plot based on WT Lyso-IP vs. control i³Neurons without HA expression. C GO enrichment analysis of significantly changed proteins in GRN KO vs. WT Lyso-IP proteomics (left: biological processes; right: molecular functions). Color code corresponds to the volcano plot. D Schematic of mouse brain Lyso-BAR labeling in GRN^−/− vs. WT mice. E Volcano plot showing Lyso-BAR protein changes in GRN^−/− vs. WT mouse brains after removing nonspecific labeling proteins. F GO enrichment analysis of significantly changed proteins in GRN ^−/− vs. WT Lyso-BAR proteomics. (G) Western blot analysis showing elevated V-ATPases and cathepsin D levels from isolated GRN KO vs. WT lysosomes in i³Neurons. H Targeted PRM protein quantification of v-ATPases and cathepsins from Lyso-BAR mouse brains. I Fluorescence imaging of Magic Red assay to measure cathepsin B enzymatic activity in i³Neurons. CQ stands for chloroquine treatment (50 μM for 24 h). Scale bar is 10 μm. J Quantification of absolute and relative fluorescence intensities indicate decreased cathepsin B activity in GRN KO vs. WT i³Neurons