Fig. 2

β-glucocerebrosidase inhibition induces substrate accumulation and reduced lysosomal activity leading to autophagic cargo accrual. a GBA1 enzymatic activity assay performed on protein extracts from undifferentiated and differentiated cells (t0 and t3) upon vehicle (veh) or CBE treatment (***, p < 0.001; Kruskal–Wallis test and Dunn's test for multiple comparison; n = 5 independent experiments). Error bars indicate s.e.m. b Immunofluorescence staining on differentiated Oli-neu cells upon vehicle (veh) or CBE treatment. Scale bar = 100 μm c Quantification of GlcCer integrated density in differentiated vehicle (veh) and CBE treated cells (*, p < 0.05; unpaired two-tailed Student’s t test; n = 11 randomly selected fields from 3 independent experiments). Error bars indicate s.e.m. d MRM-MS quantitation of Hexosylsphingosine (HexSph) in differentiated vehicle (veh) and CBE treated Oli-neu (***, p < 0.001; unpaired two-tailed Student’s t test; n = 4 independent experiments). The whiskers of the box plot represent the minimum and the maximum data values. e Representative images of LysoTracker staining of Oli-neu cells upon vehicle (veh) or CBE treatment at t3. Scale bar = 20 μm. f Quantification of LysoTracker-positive puncta in vehicle (veh) and CBE treated cells at t3 (*, p < 0.05; unpaired two-tailed Student’s t test; n = 3 independent experiments). Error bars indicate s.e.m. g Representative immunofluorescence for LAMP1 and p62 staining on Oli-neu cells upon vehicle or CBE treatment at t3. Scale bar = 20 μm. h Quantification of LAMP and p62 colocalization upon vehicle (veh) or CBE treatment in differentiated Oli-neu cells (***, p < 0.001; unpaired two-tailed Student’s t-test; n = 3 independent experiments). Error bars indicate s.e.m. i Immunofluorescence of differentiated Oli-neu cells upon vehicle (veh) or CBE treatment at t3 showing α-synuclein (α-syn) positive clusters in red. Scale bar = 50 μm. j Quantification of α-syn integrated density in differentiated vehicle (veh) and CBE treated cells (unpaired two-tailed Student’s t test; n = 9 randomly selected fields form 3 independent experiments). Error bars indicate s.e.m. k, l Western blot analysis of α-synuclein monomer (α-SYN) on differentiated Oli-neu cells treated with vehicle or CBE, and relative densitometric quantification. Vinculin (VNC) was used as loading control (*, p < 0.05; unpaired two-tailed Student’s t-test; n = 5 independent experiments). Error bars indicate s.e.m