Fig. 4

Improved trafficking and activity of cathepsins by FTI in DA-iPSn harbouring A53T mutation. a Western blot analysis of αSyn by utilizing C-20 and Syn-211 antibodies in DA-iPSn. A53T neurons were cultured with DMSO, or 5 or 10 nM FTI for 7 days with medium change every day. A53T isogenic control (iso ctrl) treated with DMSO was used as a positive control. b αSyn quantification of Western blots. Signal intensities of αSyn (detected with C-20 antibody) were normalized to the GAPDH signal and expressed as fold change, compared to A53T mutant neurons (n = 3). c CTSD activity was assessed in lysosome-enriched fractions of DA-iPSn with A53T mutation, FTI-treated A53T neurons and A53T iso ctrl (n = 3). Lysosomal activity of CTSB and CTSL was assessed in living A53T mutant neurons with or without  FTI treatment as well as A53T iso ctrl neurons (n = 4–8). d Left: Representative immunofluorescence images of A53T neurons cultured with DMSO or 5 nM FTI and respective iso ctrl. Neurons were stained for CTSD (red) and co-stained with LAMP2 (green). Nucleus is shown in blue. Scale bars: 10 µm. Right: Quantification of CTSD:LAMP2 co-staining by determining Pearson correlation coefficient, and lysosomal CTSD intensity analysis in A53T iso ctrl and A53T mutant neurons treated for 7 days with DMSO or FTI (Pearson correlation coefficient: n = 10–15 individual cells per group; lysosomal CTSD intensity analysis: n = 8–12 individual cells per group). e Left: Representative immunofluorescence images of A53T neurons cultured with DMSO or 5 nM FTI and iso ctrl. Neurons were stained for CTSB (red) and co-stained with LAMP2 (green). Nucleus is shown in blue. Scale bars: 10 µm. Right: Quantification of CTSB:LAMP2 co-staining by determining Pearson correlation coefficient, and lysosomal CTSB intensity analysis in A53T iso ctrl and A53T mutant neurons treated for 7 days with DMSO or FTI (Pearson correlation coefficient: n = 18–26 individual cells per group; lysosomal CTSB intensity analysis: n = 16–21 individual cells per group). Statistical analyses were performed by using one-way ANOVA together with a Tukey’s post-hoc test with ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05