Fig. 2

Impaired maturation and proteolytic activity of cathepsins in iPSC-derived DA midbrain neurons with pathological αSyn levels. a Representative Western blot analysis of Triton-soluble lysates of DA-iPSn of healthy control (ctrl), isogenic control (iso ctrl) and 3 × SNCA showing mature forms (heavy chain; hc) of CTSD, CTSB, and CTSL as well as αSyn. β-Actin served as a loading control. b Quantification of Triton-soluble αSyn signals (with C-20 antibody) in DA-iPSn iso ctrl, ctrl and 3 × SNCA. Signals were normalized to β-actin and expressed as fold change, compared to 3 × SNCA iso ctrl (n = 3–6). c Quantification of Triton-insoluble αSyn signals (with C-20 antibody) in DA-iPSn iso ctrl, ctrl and 3 × SNCA. Signals were normalized to β-actin and expressed as fold change, normalized to 3 × SNCA iso ctrl (n = 3). For the analysed Western blot image please see Additional file 1: Fig. S4a. d Quantifications of the hc of CTSD (left), CTSB (middle) and CTSL (right) in DA-iPSn iso ctrl, ctrl and 3 × SNCA. Each signal was normalized to the corresponding β-actin signal and shown as fold change, compared to iso ctrl (n = 3–6). e Lysosomal CTSD activity conducted from lysosome-enriched fractions (P2), and CTSL and CTSB activity assessed by live-cell activity assay in DA-iPSn iso ctrl, ctrl and 3 × SNCA (n = 3–5). f Representative Western blot analysis of αSyn levels and pro-form/single chain (sc) and heavy chain (hc) of cathepsins in A53T SNCA mutant DA-iSPn compared to the iso ctrl. β-actin signals were used to ensure equal protein loading. The asterisk (*) next to the CTSD blot marks an unspecific band. g Quantification of Triton-soluble αSyn (detected with C-20 antibody) in A53T neurons compared to iso ctrl. Signal intensity was normalized to β-actin signal and displayed as fold change (n = 4). h Quantification of Triton-insoluble αSyn (with C-20 antibody) in A53T neurons compared to iso ctrl. Signal intensity was normalized to β-actin signal and displayed as fold change (n = 3). For the analysed Western blot image please see Additional file 1: Fig. S4f. i Quantifications of the hc of CTSD (left), CTSB (middle), and CTSL (right) normalized to β-actin and expressed as fold change, compared to A53T iso ctrl (n = 4). j Western blot analysis of hc of CTSD, CTSB and CTSL in lysosome-enriched fractions of A53T and iso ctrl neurons. All three investigated cathepsins show decreased levels in A53T mutant cells compared to the iso ctrl. Statistical analyses were performed by using one-way ANOVA together with Tukey’s multiple comparison test for b–d, and two-tailed unpaired Student’s t-tests for e–i ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05