Fig. 3

ATC161 targets α-syn aggregates to p62-dependent autophagy. A Immunocytochemistry analysis in h-α-syn PFF-transduced HEK293A cells treated with ATC161 (1 μM, 12 h). B-D Quantifications of A (n = 4–5 for B and C; n = 20 cells were counted per group for the number of p62⁺-LC3⁺ colocalizing puncta in D). Data are presented as the mean (SEM). P-values (from two-sided unpaired t tests): ^* P ≤ 0.05, ^*** P < 0.001. E Western blot in MEF WT and p62 knock-out cell-lines. The cells were transduced with h-α-syn PFFs (48 h) and subsequently administered with ATC161 (1 μM, 12 h). F Western blot in MEF WT and p62 knock-out cell-lines transduced with h-α-syn PFFs (48 h) and treated with ATC161 (1 μM, 24 h). The cell lysates were subjected to Triton x-100 fractionation. G Immunocytochemistry in HEK293A cells transduced with h-α-syn PFF, followed by ATC161 treatment (1 μM, 12 h). H and I Quantifications of G (H, n = 5; I, n = 20 cells counted per group for the number of p62⁺-DFCP1⁺ colocalizing puncta). Data are presented as the mean (SEM). P-values (from two-sided unpaired t tests): ^** P-value < 0.01, ^*** P < 0.001. J Immunocytochemistry in rat cortex primary neurons transduced with h-α-syn PFFs, subsequently treated with ATC161 (1 μM, 12 h). K Quantification of J (n = 12 cells counted per group for the number of p62⁺-p-α-syn⁺ colocalizing puncta). L Proximity ligation assay (PLA) in HEK293A cells showing interaction between p62 and α-syn aggregates (with an anti-α-syn aggregate antibody). ATL7 (1 μM, 6 h), ATC161 (1 μM, 6 h), and baf. A1 (200 nM, 6 h) were administered subsequent to h-α-syn PFF transduction (48 h). M and N Quantifications from L (n = 5, n = 10, respectively), shown as the mean (SEM). P-values (from two-sided unpaired t tests): n.s. (not significant) P-value > 0.05, ^*** P-value < 0.001. O PLA image from L showing increase in puncta size by ATC161. P PLA with an anti-α-syn antibody in HEK293A cells upon transduction with recombinant h-α-syn monomers (48 h) and treatment with ATC161 (1 μM, 6 h). All the scale bars in this figure represent 10 μm