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. 2022 Aug 30;25(9):1134–1148. doi: 10.1038/s41593-022-01140-3

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© The Author(s) 2022, corrected publication 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — (a) Formation of aggregates detected by FRET is not affected by unlabeled α-Syn. (ai & ii) Representative TIRF images of labeled recombinant A53T oligomers (formed after 7 days aggregation) that were formed in the absence (ai) and presence (aii) of 7 μM unlabeled wildtype (WT) α-Syn show that the presence of unlabeled protein has no effect on aggregate formation (n = 2 independent experiments) (aiii) Size and FRET distributions of oligomers detected using single-molecule confocal microscopy when labeled α-Syn aggregates in the absence (left) or presence (right) of 7 μM unlabeled WT α-Syn. (b-d) Concentrations of endogenous α-Syn did not affect aggregation kinetics measured by single-molecule (sm) FRET. (bi & ii) Allelic series of iPSC lines display different levels of α-Syn using Human Synuclein monomer ELISA kit (bi; n = 3 independent experiments), and SNCA mRNA by qPCR (bii; average of three wells, n = 1 independent experiment). (c) A53T-AF488 and AF-594 monomers (total 1 μM) were added to the three iPSC derived neurons and smFRET was performed on the cell lysates. (ci) Analysis of total FRET (coincident) events for the iPSC lines demonstrate non-significant higher mean events for SNCA 4 alleles at 24 hours, data shown as mean ± SD. (cii & iii) 2D contour plots from smFRET measurements of cell lysates show no difference in FRET efficiencies across the three cell lines, indicating aggregates formed in cells with different levels of the endogenous α-Syn show negligibe difference in FRET efficiency. (cii) and (ciii) show two separate replicates. (di-iii) FRET measurements obtained 24 hours after A53T-AF488 and AF594 monomer treatment show there is no difference in total α-Syn uptake (dii) and FRET intensity (diii) across the cells with different endogenous SNCA expression levels (n = 3 or 4 independent experiments). Experiments were performed at an early timepoint post differentiation day 50. Note. Data are represented as Data ± SEM (box) unless mentioned. ***p < 0.0005. Detailed statistical information is provided in Supplementary Table 1.

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