Fig. 7: αSynuclein buildup enhances DOPAL-induced p62 oligomerization.

A Immunoblot of stable BE(2)-M17 cells with inducible dox-dependent αSyn overexpression, both in untreated and 100 µM DOPAL-treated for 18 h conditions. Relative quantification of B p62 monomer and C p62 oligomers after normalization to Actin used as loading control. Data are pooled together from four independent experiments normalized to each untreated sample, and analyzed by two-way ANOVA with Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, ****P < 0.0001). D Immunofluorescence of p62 (magenta) and αSyn (green) in the same cellular model. Scale bar: 20 µm and 5 µm in the inset. Relative quantification of E αSyn mean fluorescence/cell and F p62 mean fluorescence/cell. Data are pooled together from four independent experiments normalized to each untreated sample, and analyzed by two-way ANOVA with Sidak’s multiple comparison test (****P < 0.0001). G Correlation of αSyn and p62 mean fluorescence in each cell. The blue lines are centered on the reference level (normalization to 1) for each axis. On the bottom part, frequency distribution of αSyn fluorescence in overexpressing cells by 100 ng/ml dox, showing the effect of DOPAL on αSyn accumulation. On the left side of the graph, frequency distribution of p62 fluorescence in DOPAL-treated cells, showing the synergistic effect of DOPAL and αSyn overexpression on p62 buildup. H Immunoblot of non-transfected and EGFP-transfected BE(2)-M17 cells, both in untreated and 100 µM DOPAL-treated for 18 h conditions. Relative quantification of I EGFP, J p62 monomer, and K p62 oligomers after normalization to Actin used as loading control. Data are pooled together from three independent experiments normalized to each non-transfected untreated sample, and analyzed by two-way ANOVA with Tukey’s multiple comparison test (**P < 0.001, ****P < 0.0001).