Additional file 1 of The cervical lymph node contributes to peripheral inflammation related to Parkinson’s disease
Creators: name: Liu, Zongran
name: Huang, Yang
name: Wang, Xuejing
name: Li, Jia-Yi
name: Zhang, Can
name: Yang, Ying
name: Zhang, Jing
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Environmental Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Computational Biology

description: Additional file 1: Figure S1. Distribution of Qdot605 in dCLNs of A53T mice. A Representative fluorescence images of dCLNs of WT and A53T mice. Scale bar, 200 μm. B Quantitative analysis of the Qdot 605 area in dCLNs of WT and A53T mice. N = 3 independent animals in each group. Values are means ± S.E.M, t test. **, P &lt; 0.01. Figure S2. Increased inflammatory cytokines and ER stress in macrophages of dCLN of A53T mice. A Representative fluorescence images of dCLNs from WT and A53T mice treated with or without TUDCA immunolabeled by F4/80 (green) with IL-1β (red), IL-6 (red), or TNF-α (red). Scale bar, 100 μm. B Quantitative analysis of IL-1β, IL6 and TNF-α positive area in dCLNs of WT and A53T mice. N = 5 independent animals in WT and A53T. C Representative fluorescence images of GRP78 in macrophages (F4/80 positive) in dCLNs of WT and A53T mice. Scale bar, 200 μm. Values are means ± S.E.M, one-way ANOVA test. ns, not significant; ***, P &lt; 0.001; ****, P &lt; 0.0001. Figure S3. α-Synuclein in CSF was drained through the meningeal lymphatics. A Schematic image of the experimental setup for detection of lymphatic drainage path of α-synuclein in CSF. B, C Representative fluorescence images of meninges of C57 mice injected (i.c.m) with vehicle or AF647-α-synuclein (red). Scale bar, 1 mm. D, E Zoom-in images of the petrosquamous sinus in B, C. Scale bar, 200 μm. F, G Zoom-in images of the rostral rhinal vein field in B, C. Scale bar, 200 μm. H Western blot to detect α-synuclein in dCLNs of C57 mice intra-cisternally injected with vehicle, AF647-α-synuclein and α-synuclein. I Quantitative analysis of α-synuclein protein levels in dCLNs injected (i.c.m) with vehicle control, AF647-α-synuclein and α-synuclein. N = 3 independent animals in vehicle control and AF647-α-synuclein group, N = 2 independent animals in α-synuclein group. Values are means ± S.E.M, one-way ANOVA test. ns, not significant; *, P &lt; 0.05. Figure S4. α-Synuclein in CNS was drained to the macrophage in dCLN. A Representative fluorescence images of dCLNs in A53T mice immunolabeled with CD11b (green) and MJFR-1 (red). Scale bar, 200 μm. B Zoom-in image of white dotted box in A. White arrow indicates the colocalization of CD11b and α-synuclein. Scale bar, 200 μm. C Representative fluorescence images of dCLNs in A53T mice immunolabeled with CD169 (green) and MJFR-1 (red). Scale bar, 200 μm. D Zoom-in image of white dotted box in C. White arrow indicates the colocalization of CD169 and α-synuclein. Scale bar, 200 μm. E, F Quantitative analysis of the levels (pg/mg) of total α-synuclein and α-synuclein oligomer in macrophages of dCLN and total dCLN lysate using MSD, normalized by total protein concentration. N = 3 independent sample pools in each group. Values are means ± S.E.M, t test. *, P &lt; 0.05. Figure S5. Peripheral inflammation in A53T mice. A Volcano plot of the significantly up- and down-regulated genes in the dCLNs of WT and A53T mice. N = 3 independent in each group (3 WT samples were pooled from 6 animals; 3 A53T samples were pooled from 4 animals). P values (Padj, adjusted P values) were corrected for multiple hypothesis testing with the Benjamini–Hochberg false discovery rate procedure. B Principal component (PC) analysis of the transcriptome of dCLNs of WT and A53T mice. N = 3 independent in each group (3 WT samples were pooled from 6 animals; 3 A53T samples were pooled from 4 animals). C The scatter diagram of GO enrichment of differentially expressed genes in dCLNs of WT and A53T mice. D Quantitative analysis of the mRNA level of typical genes in ER stress, including Ern1, Xbp1, Atf6, Atf4, Ddit3, and Hspa5. N = 3 independent animals in each group. Values are means ± S.E.M, t test. *, P &lt; 0.05. Figure S6. Heat map of genes involved in response to endoplasmic reticulum stress signaling pathway. Red represents gene expression levels above the mean; blue represents gene expression levels below the mean. N = 3 independent in each group (3 WT samples were pooled from 6 animals; 3 A53T samples were pooled from 4 animals). Figure S7. α-Synuclein monomer elicited ER stress weakly in macrophages. A Western blot to assess the levels of p-IRE1α, IRE1α, GRP78, ATF6, ATF6-p, ATF4, sXBP-1, p-eIF2α, eIF2α and α-synuclein in BMDMs treated with vehicle control, 100 nM or 400 nM α-synuclein monomer. B–J Quantitative analysis of the levels of p-IRE1α, IRE1α, RP78, ATF6, ATF6-p, ATF4, sXBP-1, p-eIF2α and eIF2α in BMDMs treated with vehicle control, 100 nM or 400 nM α-synuclein monomer. N = 3 independent experiments in each group. K Quantitative analysis of Ddit-3 mRNA levels in BMDMs treated with vehicle control, 100 nM or 400 nM α-synuclein monomer using qPCR. N = 3 independent experiments in each group. Values are means ± S.E.M, one-way ANOVA test. ns, not significant; *, P &lt; 0.05; **, P &lt; 0.01; ***, P &lt; 0.001. Figure S8. α-Synuclein aggregate elicited ER stress in the dCLN. A Schematic image of the experimental setup to verify whether α-synuclein oligomer can elicit ER stress in the dCLN. B Western blot to assess the levels of p-IRE1α, IRE1α, GRP78, ATF6, ATF6-p, p-eIF2α, and eIF2α in dCLNs of C57 mice treated with vehicle control or 2 μg α-synuclein oligomer. C–I Quantitative analysis of the levels of p-IRE1α, IRE1α, GRP78, ATF6, ATF6-p, p-eIF2α and eIF2α in dCLNs of C57 mice treated with vehicle control or 2 μg α-synuclein oligomer. N = 3 independent experiments in each group (3 samples were pooled from 6 animals). Values are means ± S.E.M, one-way ANOVA test. ns, not significant; *, P &lt; 0.05; **, P &lt; 0.01. Figure S9. TUDCA improved the motor function and rescued neuronal death of A53T mice. A The latency to fall off from the rod for WT and A53T mice treated with or without TUDCA. N = 5 independent animals in each group. B The time taken for the mice to land from the top for WT and A53T mice treated with or without TUDCA in the pole test. N = 5 independent animals in each group. C, D Immunohistochemical staining and quantification of tyrosine hydroxylase (TH) positive neurons in the substantia nigra of WT and A53T mice treated with or without TUDCA. N = 3 independent animals in each group. Values are means ± S.E.M, one-way ANOVA test. ns, not significant; *, P &lt; 0.05; **, P &lt; 0.01; ***, P &lt; 0.005, ****, P &lt; 0.001. Table S1. Numbers of animals per study group.