Skip to main content
. 2021 Apr 8;118(15):e2025847118. doi: 10.1073/pnas.2025847118

Fig. 2.

Fig. 2.

Oligomeric αSyn provides two signals for NLRP3 inflammasome activation. (A) Dynamic light scattering analysis of the particle size of the two types of αSyn preparations used in this study—αSyn monomers and oligomer/aggregates. Fractions of monomer vs. aggregated αSyn categorized by hydrodynamic diameter (D) on DLS are shown in different shading. Two-way ANOVA followed by Sidak’s multiple-comparisons test revealed a significant difference between the two preparations for monomeric αSyn as well as aggregates >2,000 nm. No significant difference was found for aggregates of 10 to 200 and 200 to 2,000 nm (light gray and dark gray bars). (B) Quantification of IL-1β secretion following inflammasome activation with LPS + ATP/Alum/Nigericin (n = 5 per group). (C) IL-1β secretion following exposure to αSyn monomers and oligomers (750 nM, n = 10 per group). (D) Cleaved caspase-1 (p20) release following exposure to αSyn monomers and oligomer (750 nM, n = 5 to 9). (E) IL-1β secretion following NLRP3 knockdown and exposure to oligomeric αSyn (750 nM, n = 5 per group). (F) Quantification of IL-1β following treatment with neutralizing antibodies against TLR2 and TLR4 or controls (IgA and IgG, respectively) (n = 6 per group). (G) Representative images of MitoSOX fluorescence reflecting mitochondria-generated ROS following vehicle (control) and monomer and oligomer αSyn exposure (750 nM). (Scale bars, 100 µm.) (H) Quantification of MitoSOX fluorescence after exposure to monomeric or oligomeric αSyn (750 nM, n = 9 per group). (I) Representative images of tetramethylrhodamine methylester (TMRM) fluorescence, assessing mitochondrial membrane potential, following control and monomer and oligomer αSyn exposure (750 nM). (Scale bars, 200 µm.) (J) TMRM quantification after exposure to monomeric or oligomeric αSyn (750 nM, n = 5 to 10). (K) mtDNA release of ND5, ND6, and COX1 following exposure to αSyn monomers and oligomers (750 nM, n = 3 to 5). Graphs indicate mean ± SEM. Statistical analysis was performed using two-way ANOVA with Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).