Figure 2.

DMSO stimulated fibrils resemble naïve α-syn fibrils and maintain seeding capabilities (A) Representative image of TEM shows structure of wt α-syn PFFs or α-syn DMSO PFFs made in the presence of 2% DMSO. Scalebar = 200 nm. Representative images of three replicates (n = 3). (B) Digest samples of 25.000×g pelleted wt α-syn- and α-syn DMSO PFFs. Samples were digested with depicted concentrations of Proteinase K (PK). Digested samples were resolved on SDS-PAGE and stained at RT, using Coommassie Brilliant Blue. Representative gel of three independent replicates (n = 3). (C) Viability of α-syn-expressing SHSY5Y ASYN cells was measured by MTT assay 8 days post seeding. On day 2 the cells were left untreated (control) or treated with PBS (+ PBS), α-syn PFFs (28 μg/mL/2 μM) or 2% DMSO α-syn PFFs (28 μg/mL/2 μM) which was removed on day 4. Bars represent relative viability normalized to control. Three biological replicates with 10 measurements in each experiment (n = 3, ****p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test) (D) Human a-syn expressing OLN-AS7 cells were exposed to PBS (Control) or 14 μg/mL (1 μM) sonicated S129A α-syn PFFs prepared in PBS alone (S129A α-syn PFFs) or in the presence of 2% DMSO (2% DMSO S129A α-syn PFFs). After 24 h of PFF treatment, the cells were washed to remove excess PFF and subsequently incubated for another 24 h before being fixed and visualized using DAPI (blue), α-tubulin (purple), total α-syn (red) or anti-phospho-S129 α-syn (green). Scale bar = 20 µm. Representative images of three biological replicates with 10 measurements in each experiment. (E) Quantification of area of anti-phospho-S129 α-syn signal relative to number of DAPI stained nuclei. Data are shown as mean of three independent experiments (n = 3, ****p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test).