Fig. 3

Farnesyltransferase inhibitor (FTI) treatment decreases αSyn level and ameliorates impaired cathepsins trafficking in H4 cells overexpressing αSyn. a Schematic illustration of FTI (LNK-754) mechanism inside the cell. The application of the compound induces a lysosomal stress pathway, which boosts hydrolase trafficking towards the lysosome. b Western blot analysis of αSyn using C-20 and Syn-211 antibodies in H4 cells cultured with 5 nM or 10 nM FTI for 5 days with every day medium change. DOX treatment was used as a positive control to show low αSyn levels. GAPDH and CBB served as loading controls. c Corresponding quantification of αSyn signals detected with C-20 αSyn antibody in H4 cells, which were normalized to GAPDH and shown as fold change, compared to high αSyn (DMSO) (n = 4–5). d Lysosomal CTSD activity of lysosome-enriched fraction and live-cell lysosomal CTSL and CTSB activity assay of H4 cells overexpressing αSyn (DMSO), with and without FTI treatment (5 days treatment with every day medium change, n = 3–5). e Left: Representative immunofluorescence images of αSyn-overexpressing (DMSO-treated) H4 cells with or without 5 nM FTI treatment, stained for CTSD (red) and co-stained with LAMP2 (green). Nucleus is stained with DAPI. Scale bars: 10 µm. Right: Pearson correlation coefficient analysis of CTSD:LAMP2 co-staining, and lysosomal CTSD intensity analysis in H4 cells with or without 5 nM FTI treatment (Pearson correlation coefficient: n = 10–11 individual cells per group; lysosomal CTSD intensity analysis: n = 10–12 individual cells per group). f Left: representative immunofluorescence images of αSyn-overexpressing (DMSO-treated) H4 cells with or without 5 nM FTI treatment, stained for CTSB (red) and co-stained with LAMP2 (green). Nucleus is stained with DAPI. Scale bars: 10 µm. Right: Pearson correlation coefficient analysis of CTSB:LAMP2 co-staining, and lysosomal CTSB intensity analysis in H4 cells with or without 5 nM FTI treatment (Pearson correlation coefficient: n = 24–26 individual cells per group; lysosomal CTSB intensity analysis: n = 10 individual cells per group). Statistical analyses were performed by using one-way ANOVA together with Dunnett’s post-hoc test for c, and two-tailed unpaired Student’s t-tests for d–f. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05