Supplementary Figure 2: Analysis of lysosome-enriched fractions from WT and CLN8-deficient mice.

(a), Lysosome-enriched fractions were harvested upon liver homogenization and ultra-centrifugation in a discontinuous Nycodenz gradient. (b), HEXA activity assay showed significant enrichment for lysosomes in the collected fractions and no obvious changes in activity in the comparison of WT and Cln8^mnd mice. (c), LAMP1 immunoblot showing lysosomal enrichment in collected fractions and no obvious changes between WT and Cln8^mnd samples. (d), Details of the plot of relative protein signals from LC-MS/MS analysis of lysosome-enriched liver fractions from WT and CLN8-deficient mice (KO). Each blue dot (representing soluble lysosomal enzymes) has been labeled with the corresponding protein symbol; red dots and grey dots represent lysosomal membrane proteins and other proteins detected by LC-MS/MS, respectively. Data are relative to n = 2 independent proteomic analyses conducted on pools of three livers each. (e), GSEA of lysosomal membrane proteins and mitochondrial matrix proteins in the comparison of lysosome-enriched fractions from the livers of WT and CLN8-deficient mice. Proteins detected by LC-MS/MS are ranked according to their enrichment in CLN8-deficient vs. WT mice (left: increased in CLN8-deficient mice, red; right: decreased in CLN8-deficient mice, blue). GSEA shows that the sets of proteins are not differentially distributed in CLN8-deficient vs. WT mice (P_{lysosomal membrane proteins} = 0.31; P_{mitochondrial matrix proteins} = 0.32). (f), Enzymatic assay of GAA, GALNS, GBA, GLB1 and TPP1 in liver lysosome-enriched fractions from 2-month-old WT and Cln8^mnd mice. Activity is expressed as percentage of the WT samples. All enzyme activities were conducted by using fluorophore analogs of enzymes substrates, as previously described: TPP1¹; GAA²; GBA³; GLB1⁴; GALNS⁵. (g), Expression analysis of lysosomal genes in the liver of 2-month-old WT and Cln8^mnd mice. Shown is the fold-change in expression of genes in the Cln8^mnd relative to WT mice. Expression levels are normalized to the housekeeping gene Ppif (peptidylpropyl isomerase F or cyclophilin D). Images shown in (a) and (c) are representative of n = 3 independent experiments. In (b), (f) and (g), data are means ± SEM (n = 3 independent experiments, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test)