Fig 2: TRPM2 knockout reduces the expression of multiple atherosclerosis-related proteins in the plaque region of aortic roots. (A) Representative tissue-section images of immunohistochemical stains of CD68 (upper), a-SMA (middle), and PCNA (lower) in aortic roots from TRPM2+/+ and TRPM2-/- mice fed with a high-cholesterol diet for two months. (B) Representative tissue-section images of immunohistochemical stains of ICAM-1 (upper), MCP-1 (middle), and TNFa (lower) in aortic roots from TRPM2+/+ and TRPM2-/- mice fed with a high-cholesterol diet for two months. Brown color represents immune-positive signals in lesion area. Blue color represents hematoxylin counterstaining of cell nuclei. For all experiments, shown are representative images from four mice. Scale bar, 100 µm. (C,D) Quantification of immune-positive signals in (A,B), expressed as integrated optical density (IOD) divided by area. Mean ± SD (n = 4). (E) qRT-PCR quantification for the expression of CD68, a-SMA, PCNA, ICAM-1, MCP-1, and TNFa in whole-aorta samples in atherosclerotic mice fed with a high cholesterol-diet for two months. Mean ± SEM (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 3: Inflammatory responses of microglia treated with two a-syn preparations. Inflammatory responses of primary human brain microglia when treated with vehicle, ribbons, fibrils (100 nM), LPS (10 ng/mL) and IL-1ß (10 ng/mL). (A) Representative immunocytochemistry images of ICAM-1 induction after 48 h treatment Scale bar = 200 µm. Immunocytochemistry quantification of (B) NF-?B nuclear translocation after 1 h treatment, (C) MCP-1 and (D) ICAM-1 induction after times indicated (n = 3, mean ± SD). Microglia were cultured and treated with vehicle, two different a-synuclein preparations (100 nM), LPS (10 ng/mL) and IL-1ß (10 ng/mL) before conditioned media were taken and cytokine secretion analysed by cytometric bead array. Heatmap analysis of cytokine secretion of microglia after treatment at (E) 8 h, (F) 24 h and (G) 48 h (n = 3). Microglia were cultured and treated with vehicle, two different a-synuclein preparations (100 nM), LPS (10 ng/mL) and IL-1ß (10 ng/mL) for 24 h before conditioned media were taken and cytokine secretion measured using a Proteome Profiler Human XL Cytokine Array Kit. Clustered heatmap analysis (H) of the top 30 secretions in microglia (n = 1, epilepsy case). #LPS, + IL-1ß vs vehicle p < 0.05; one way ANOVA with Tukey’s multiple comparisons test or two way ANOVA with Tukey’s multiple comparisons test.

Fig 4: AM reduces the expression of genes involved in inflammation. Gene expression analysis of VCAM-1 (A), ICAM-1 (B), CCL2 (C), and SELE (D), from C- and GD-HUVECs untreated and after TNF-a stimulation of cells that had been pretreated or not with AM for 24 h. Quantitative data in the histograms (A–D) show the mRNA relative expression (target gene expression normalized with GAPDH expression using the 2-??Ct method). Each measurement is defined as the mean ± SD using three different strains for C-HUVECs and three different strains for GD-HUVECs (n = 3). ANOVA and Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 5: TLR ligands and a-syn treatment on pericytes. Inflammatory responses of primary human brain pericytes when treated with vehicle, ribbons (100 nM), fibrils (100 nM) and TLR ligands (Supplementary Table S5). Immunocytochemistry quantification of (a) NF-?B nuclear translocation after 1 h treatment. Conditioned media were taken and cytokine secretion analysed by cytometric bead array after 24 h. Secretions of (b) ICAM-1, (c) VCAM-1, (d) Fractalkine, (e) IL-6, (f) IL-8, (g) MCP-1, (h) IP-10 and (i) RANTES at 24 h after treatment (n = 5 epilepsy cases, mean ± SD).