Fig. 6: Progranulin-boosting drugs correct aberrant lysosomal proteome.
A, B Western blot validation demonstrates processed TPP1 levels in GRN+/− HDFs is reduced, and 24-h treatment with 50 µM A41 upregulates TPP1 levels (n = 4 GRN+/+, n = 5 GRN+/−, n = 5 GRN+/− + A41) (*p = 0.03; **p = 0.005 two-sided Student’s t-Test). C RT-qPCR demonstrates upregulation of GRN, TPP1, and DPP7 levels by 24- and 72 h treatment with 50 µM A41 (n = 5 GRN+/+, n = 6 GRN+/− + A41 24’, n = 5 GRN+/− + A41 72’) (**p < 0.01; ***p < 0.001; One-way ANOVA, Holm-Å Ãdák correction). D Representative fluorescent microscopy images of TFEB-GFP (green) and DAPI (blue) in 293 T cells treated with various concentrations of A41 or EBSS (positive control). Micrographs represent 3 independent experiments. E Quantification of TFEB-GFP nuclear/cytoplasmic ratios (*p = 0.014; ****p = 1.0 × 10−15; One-way ANOVA, Holm-Å Ãdák correction). F Volcano plot of lysosomal proteomes from Grn+/− MEFs treated for 24 h with 100 µM A41 compared to vehicle. G PCA depicting variations between DMSO treated Grn+/+, DMSO or A41 treated Grn+/−, and DMSO treated Grn−/− MEF lysosomal proteomes. H Volcano plot of lysosomal proteomes from GRN+/+ HDFs treated for 72 h with 50 µM A41 compared to vehicle. I PCA depicting variations between DMSO treated GRN+/+ and DMSO or A41 treated GRN+/− HDF lysosomal proteomes. J A41 treatment corrects the levels of several key lysosomal proteins in Grn+/− MEF lysosomes (n = 6 Grn+/−, n = 6 untreated Grn+/−, n = 3 A41 treated Grn+/−) (*q < 0.05; Two-stage step-up Benjamini, Krieger, and Yekutieli Test). K A41 treatment rescues the lysosomal expression of several proteins reduced in GRN+/− HDFs (n = 5 GRN+/−, n = 6 untreated GRN+/−, n = 5 A41 treated GRN+/−) (*q < 0.05; Two-stage step-up Benjamini, Krieger, and Yekutieli Test). All error bars represent standard error of the mean.
