Additional file 1 of Multi-modal proteomic characterization of lysosomal function and proteostasis in progranulin-deficient neurons
Creators: name: Hasan, Saadia
name: Fernandopulle, Michael S.
name: Humble, Stewart W.
name: Frankenfield, Ashley M.
name: Li, Haorong
name: Prestil, Ryan
name: Johnson, Kory R.
name: Ryan, Brent J.
name: Wade-Martins, Richard
name: Ward, Michael E.
name: Hao, Ling
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Biological Sciences not elsewhere classified
subject: Mental Health

description: Additional file 1: Supplementary Figure S1. Confirmation of the Lyso-IP, Lyso-BAR, and Lyso-APEX probe locations at the lysosome, related to Figure 1. (A) Fluorescence imaging of Lyso-APEX i3Neurons and negative control neurons without H2O2 treatment. Biotinylation cloud stained with streptavidin (SA-680), colocalizes with the bait protein LAMP1. Negative control group does not have SA signal. Nuclei were stained by Hoechst. Scale bar is 10 μm. (B) Western blot analysis of isolated lysosomes from Lyso-IP i3Neurons compared to whole cell lysate and negative control without HA expression. (C) Protein network analysis of Lyso-IP enriched known lysosome proteins from i3Neurons. (D) Protein network analysis showing enriched lysosome, vesicle-mediated transport, and synapse processes from Lyso-Bar enriched proteins in mouse brains compared to Lyso-APEX enriched proteins in i3Neurons. (E) Venn diagram comparison of significantly enriched proteins in Lyso-APEX, Lyso-IP, and Lyso-BAR proteomics. Supplementary Figure S2. Proximity labeling proteomics and lysosome pH measurement in progranulin-null i3Neurons, related to Figure 2. (A) GO enrichment analysis of significantly upregulated molecular functions in GRN KO vs. WT Lyso-APEX proteomics. (B) GO enrichment analysis of significantly downregulated molecular functions in GRN KO vs. WT Lyso-APEX proteomics. (C) Volcano plot of cytosolic-APEX proteomics in GRN KO vs. WT i3Neurons (N=4 for each group). (D) Scatter plot of significantly changed proteins (corrected p-value &lt;0.05) in both Lyso-APEX and cytosolic-APEX proteomics showing potential protein translocation in neurons. (E) Lysosomal pH measurements in WT vs. GRN KO i3Neurons with linear (left) and 3rd order (right) calibration curve fitting. Supplementary Figure S3. Loss of progranulin results in elevated levels of lysosomal catabolic enzymes and decreased cathepsin B activity in human i3Neurons and mouse brains, related to Figure 3. (A) PGRN is enrichment in isolated WT lysosomes and is absent in GRN KO lysosomes from Lyso-IP proteomics data. (B) Additional replicate of Magic Red assay showing reduced cathepsin B activity in GRN KO i3Neurons compared to WT. Scale bar is 10 μm. (C) Volcano plot of Lyso-IP proteomics in KO vs. WT i3Neurons without filtering, related to Figure 3B (N=4 for each group). (D) Protein network analysis of selected lysosome and synaptic proteins that are significantly changed in GRN KO vs. WT Lyso-IP proteomic. (E) Volcano plot of Lyso-BAR proteomics in GRN-/- vs. WT mouse brains without filtering, related to Figure 3E (N=4 for each group). (F) Protein network analysis of selected lysosome and synaptic proteins that are significantly changed in GRN-/- vs. WT Lyso-BAR proteomics. Supplementary Figure S4. Developing dynamic SILAC proteomics in i3Neurons to measure global neuron protein half-lives, related to Figure 4. (A) Histogram distribution of peptide level curve fitting R2 to first-order exponential decay. An example of Tau peptide degradation and synthesis curves is shown in the inset. (B) Scatter plot of ranked peptide level half-lives in WT i3Neurons. (C) Histogram distribution of peptide half-lives, consistent with protein level results in Figure 4D. (D) Scatter plot showing strong correlation of protein/peptide half-lives measured by multiple-time-point method (1, 2, 4, 6 days) and single-time-point method (4 day). Supplementary Figure S5. DQ-BSA Red Assay to measure lysosomal degradative function, related to Figure 5. (A) Chloroquine treatment (30 µM for 12 hours) impairs the lysosomal degradative function with significantly reduced DQ-BSA signals compared to untreated WT i3Neurons (*** denotes p-value &lt; 0.001). (B) WT and GRN KO i3Neurons have similar total number of DQ-BSA fluorescent puncta, indicating similar overall levels of endocytosis and lysosomal biogenesis, related to Figure 5I. Supplementary Figure S6. Protein half-life changes caused by progranulin deficiency in GRN-KO, WT, ptKO, ptMut, and ptWT i3Neurons, related to Figure 6. (A) Scatter plot showing potential gene dosage effect of protein half-life changes in ptKO vs. ptWT and ptMutant vs. ptWT i3Neurons. (B) Heatmap showing overlapping protein half-lives in WT, GRN-KO, ptWT, ptKO, and ptMut i3Neurons. Heatmap colors represent the absolute half-life measurements in days.