Fig 2: The lysosomal network protein LC3 is upregulated in the CSF of clinically diagnosed 4-repeat tauopathy patients. A) Representative samples from one Western blot of CSF from controls (C) and clinically diagnosed 4-repeat tauopathy patients (CBS/PSP), analyzed for the lysosomal network proteins EEA1, LAMP-1, LAMP-2, LC3 and lysozyme B) Mean densitometric quantification of the scanned Western blots from C (n = 11), clinically diagnosed 4-repeat tauopathy patients (CBS, n = 6 and PSP, n = 5). The protein levels are normalized to a standard CSF sample loaded on each gel. C) Equal sample loading was verified by Ponceau S staining of total protein in each lane on the membranes. The bars represent the mean±SD, ***p < 0.001.

Fig 3: The lysosomal network proteins LAMP-1, LAMP-2, LC3 and lysozyme are upregulated in the CSF of pathologically confirmed CBD patients, whilst EEA1 is decreased and lysozyme is upregulated in the CSF of pathologically confirmed PSP patients. A) Representative samples from one Western blot of CSF from controls (C) and pathologically confirmed corticobasal degeneration (CBD) patients analyzed for the lysosomal network proteins EEA1, LAMP-1, LAMP-2, LC3 and lysozyme. B) Mean densitometric quantification of the scanned Western blots from C (n = 10) and pathologically confirmed CBD (n = 7) patients. C) Representative Western blots of CSF from controls (C) and pathologically confirmed progressive supranuclear palsy patients (PSP) analyzed for the lysosomal network proteins EEA1, LAMP-1, LAMP-2, LC3 and lysozyme. D) Densitometric quantification of the scanned Western blots from C (n = 10) and pathologically confirmed PSP patients (n = 8). The protein levels are normalized to a standard CSF sample loaded on each gel. E and F) Equal sample loading was verified by Ponceau S staining of total protein in each lane on the membranes in A and C respectively. The bars represent the mean±SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: LLOMe-induced apoptosis is preceded by release of lysosomal constituents to the cytosol.Human fibroblasts were exposed to 0–1 mM LLOMe for selected time points. a Immunostaining of galectin-3 (green) and DAPI (blue) with b corresponding quantitative analysis of cells positive for punctuate galectin-3 staining after exposure to 1 mM LLOMe. Lysosomes in >50 cells from randomly selected areas were quantified (n = 4). c Immunostaining of galectin-3 (green), LAMP2a (red) and DAPI (blue) in cells exposed to 1 mM LLOMe. d Enzymatic activity of N-acetyl-ß-d-glucosaminidase in cytosolic extracts (n = 3). e Cathepsin B activity in cytosolic extracts (n = 3). f Immunoblot of cell fractions obtained by differential centrifugation from cells exposed to 1 mM LLOMe for 2 h. LDH is used as a cytosolic marker, and cathepsin D as an indicator of LMP. g Immunoblot of LAMP2 in digitonin-extracted cytosolic fractions obtained from fibroblasts pretreated with Z-VAD-FMK (10 µM, 1 h) followed by 1 mM LLOMe for 2 h with corresponding quantitative analysis (n = 4). *p < 0.05, **p < 0.01 and ***p < 0.001 compared to untreated control.

Fig 5: Analysis of endolysosomal compartments (related to Figs 4 and 5) AQuantification of cellular uptake of fluorescently labeled transferrin in fibroblast lysates, normalized to total protein levels (n = 3 biological replicates). Data represented as mean ±SEM. Statistical comparisons by unpaired two-tailed Student’s t-test (n = 3; P = 0.0023). **P < 0.01.BImmunoblot (left) of RAB11B in the indicated fibroblasts, under basal and serum-starved conditions, and (right) summary quantifications normalized to levels of actin (ACTB) and expressed as % of controls. Bar graphs represent data as mean ±SEM (n = 3 biological replicates).CRepresentative confocal micrographs of fibroblasts stained for LAMP2 and LysoTracker. Scale bar 10 µm.DQuantification of the co-localization between LysoTracker and LAMP2, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n = 10 optical sections from three independent experiments).EQuantification of the number and intensities of LAMP2-stained puncta. Data represented as mean ± SEM (n = 6).