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. 2023 May 29;29(6):1448–1455. doi: 10.1038/s41591-023-02358-9

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© The Author(s) 2023

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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Fig. 3 — a, Intracellular α-synuclein inclusions generated by serum IP/RT-QuIC products derived from patients with PD, MSA and DLB are visualized using SR-SIM. Representative morphologies of GFP-fused α-synuclein A53T inclusions generated by each disease-derived seed transduction in 293-SynG cells are shown. There are three different morphologies, including fibrous (FL), dense-core (DC) and pale-core (PC) inclusion (n = 5 different samples). The inset shows a single layer of the center slice of the inclusions. Scale bars are 2 μm, and the inset sides of the square are 10 µm. b, Images using BZ-X810 are generated as the full-focus image based on 10–20 z-stack images with 3-µm steps with a ×40 objective lens, and insets represent magnified images of inclusions in the square. Representative low-resolution images of 293-SynG cells transduced with each disease-derived seed. c, Violin plots indicate the ratio of each type of inclusion in five (MSA and DLB/PDD) or six (PD) independent low-resolution images obtained from each disease-specific seed transduced cell and evaluated in a blinded manner by three independent examiners as indicated. Statistical analysis was performed using a two-sided one-way ANOVA followed by Tukey’s correction, resulting in significances of P < 0.05 (*), P < 0.001 (***) and P < 0.0001 (****) among FL, DC, PC and ND among each synucleinopathy (PD: FL versus DC, P < 0.0001; FL versus PC, P < 0.0001; DC versus PC, P = 0.9596; MSA: FL versus DC, P = 0.0002; FL versus PC, P = 0.0131; DC versus PC, P = 0.2351; DLB/PDD: FL versus DC, P = 0.3194; FL versus PC, P < 0.0001; DC versus PC, P < 0.0001). d, The fluorescence density of intracellular α-synuclein inclusions, which was calculated as low-resolution fluorescence intensity of inclusion bodies divided by the area of inclusions, generated by seeds derived from patients with PD, MSA and DLB/PDD (PD versus MSA, P < 0.0001; PD versus DLB/PDD, P < 0.0001; MSA versus DLB/PDD, P = 0.0254) (n > 20 for each group). Violin plots show the range and average distribution. Statistical analysis was performed using a two-sided one-way ANOVA followed by Tukey’s correction, resulting in significances of P < 0.0001 (****) and P < 0.05 (*) among PD, MSA and DLB/PDD. ND, not determined; NS, not significant.