Fig. 3

Co-localization of PFF-induced ps-αSyn with mitochondria marker TOM20. a Primary cortical neurons were prepared from OVX mice, cultured for 7 days, then treated with hPFF (PFF prepared with human αSyn), cultured for another 9 d, and subjected to immunofluorescence staining with antibodies against ps-αSyn and TOM20. Inserts are enlarged images of the areas indicated by the dashed boxes. b Mouse primary neurons were treated with PFF and subjected to immunofluorescence staining with antibodies against ps-αSyn and TOM20. Arrows point to the area with strong ps-αSyn accumulation, but weak TOM20 staining. The Pearson correlation coefficient in a and b was determined from 12 individual cells. Statistic difference was determined by an unpaired t-test (n = 12; p < 0.0001). c A proximity ligation assay (PLA) with antibodies against ps-αSyn and TOM20 was performed in primary neurons treated with αSyn monomer or PFF. Representative images include left, negative control (omitting anti-TOM20 antibody); center, αSyn monomer-treated neurons; right, PFF-treated neurons. The bar graph shows average ± standard error of three independent experiments with six areas quantified per experiment. Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparisons test (F = 66.40; n = 6; p < 0.0001)