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. 2024 Jul 4;10:129. doi: 10.1038/s41531-024-00738-7

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a and b Effect of pretreatment on varied temperature on the seeding ability of human pre-formed fibrils (hPFFs)/ mouse pre-formed fibrils (mPFFs). 1 μg/ml hPFFs/mPFFs were exposed to heating (121, 70 °C) or cooling (−20, −80 °C) for 1 h before being utilized in the streamlined SAA. c Transmission electron microscopy (TEM) images of hPFFs/mPFFs after cooling pretreatment at −20 °C for 72 h. The hPFFs depolymerized into 20 nm diameter oligomers, while the mPFFs showed no significant depolymerization compared to hPFFs. Scale bar = 200 nm. d Immunofluorescence image of pS129-Syn in primary neurons exposed to heating (121 °C) or cooling (−20 °C) pretreated hPFFs (1 μg/ml) for ten days, untreated hPFFs were used as control. Scale bar = 25 μm. e and f Effect of grinding pretreatment on the seeding ability of hPFFs/mPFFs. 1 μg/ml hPFFs/mPFFs were pretreated by grinding for either 0, 1, or 10 times before being utilized in SAA. The decreased seeding capacity from over-grinding (10 times) was reversed by the extra addition of 0.1 mg/ml of homologous monomer to the grinding solution. One-way ANOVA followed by Tukey’s post-hoc-test was used, data are shown as mean ± SD. Data are statistically different from each other with *P < 0.05, **P < 0.01, and ***P < 0.001.