Additional file 6: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S4. Seeding activities of serial dilutions of treated synthetic α-syn fibrils in SH-SY5Y cells, Serial dilutions of synthetic α-syn fibrils exposed to various inactivation treatments were introduced into SH-SY5Y cells. Immunoblot analysis of sarkosyl-insoluble fractions (ppt) and sarkosyl-soluble fractions (sup) extracted from cells transfected with serial dilutions of synthetic α-syn fibrils treated with 1% SDS for 1 h at room temperature, boiling, or autoclaving at 134 °C with or without 1% SDS are shown. Phosphorylated α-syn was detected with anti-phosphorylated α-syn PSer129 antibody. α-Syn was detected with anti-syn 131–140 antibody. (PDF 248 kb

Additional file 2: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Table S1. α-Syn concentrations in sarkosyl-insoluble fractions extracted from patients’ brains, The α-syn concentrations of sarkosyl-insoluble fractions extracted from patients’ brains used for experiments in the cultured cell model (A) and mouse model (B) are shown. (PDF 40 kb

Additional file 4: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S3. Determination of protein concentration of α-syn in C57BL/6 mouse brain, Standard curve of mouse α-syn was generated by immunoblotting of serial dilutions of recombinant mouse α-syn protein. Protein concentrations of endogenous α-syn in mouse brains were determined by interpolation on a standard curve. A68 buffer-soluble fractions were extracted from C57BL/6 mouse brains (n = 3). Bands of recombinant proteins and A68 buffer-soluble fractions were detected with anti-mouse α-syn antibody. (PDF 78 kb

Additional file 1: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S1. Determination of protein concentration of phosphorylated α-syn in patients’ brains. A, Sarkosyl-insoluble fractions prepared from patients’ brains used in this study were analyzed by immunoblotting with anti-phosphorylated α-syn PSer129 antibody (upper) and anti-tau T46 antibody (lower). B, Standard curve of phosphorylated α-syn, generated by immunoblotting of phosphorylated monomer α-syn. Concentrations of phosphorylated α-syn were determined using this standard curve. Protein concentrations of sarkosyl-insoluble fractions extracted from patients’ brains are shown in Table S2. (PDF 139 kb

Additional file 5: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Table S2. Numbers of mice used in experiments. (PDF 52 kb

Additional file 3: of Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

Figure S2. Seeding activities of serial dilutions of sarkosyl-insoluble fractions from brains of α-synucleinopathy patients, Sarkosyl-insoluble fractions extracted from brains with synucleinopathy patients were diluted and introduced into SH-SY5Y cells transiently expressing human α-syn. Immunoblot analyses of sarkosyl-insoluble fractions (ppt) and sarkosyl-soluble fractions (sup) extracted from cells transfected with serial dilutions of MSA-1(Cb), MSA-2 (FC), MSA-2 (Pu), MSA-3 (FC) and DLB-4 (FC) are shown. Phosphorylated α-syn was detected with anti-phosphorylated α-syn PSer129 antibody. α-Syn was detected with anti-syn 131–140 antibody. Cb: cerebellum, FC: frontal cortex, Pu: putamen. (PDF 283 kb

Densities of olig2^weak(+) and olig2^strong(+) nuclei in 1 mg of gray matter tissue from BPD FPC. (A) olig2^weak(+), and (B) olig2^strong(+) nuclei.

<p>*<i>P</i><0.05 by unpaired <i>t</i>-test.</p

Densities of nuclei in 1 mg of gray matter tissue.

<p>(a-d) FPC and (e-h) ITC. (a, e) 7-AAD(+), (b, f) NeuN(+), (c, g) olig2(+), and (d, h) NeuN(−)/olig2(−) nuclei. *P<0.05 by unpaired <i>t</i>-test.</p

Validation of forward scatter (FS) values of nuclei isolated from frozen unfixed human postmortem brains.

<p>(A) Appearance of a human NeuN(+) (upper) or olig2(+) nucleus (lower) in PBS (280 mOsm). Scale bar, 20 µm. (B) FCM measurement of 7-AAD(+) nuclei. Arrows indicate two major populations of 7-AAD(+) nuclei. (C) Stability of isolated NeuN(+) nuclei in PBS. No difference was found in the FS distributions of NeuN(+) nuclei analyzed at 4 h and 18 h after homogenization, suggesting that the size of NeuN(+) nuclei remained quite stable in PBS. (D) Distribution of 7-AAD(+) (red) and NeuN(+) (green) nuclei areas (µ m²) as determined by microscopic measurements. The nuclei populations were delineated every 50 µm². (E) Correlation between the areas of 7-AAD(+) and NeuN(+) nuclei (µ m²) as measured microscopically. The arrow (200 µm² of NeuN(+) area) indicates a point that corresponds approximately to FS 400. Beginning with this point, the area of the NeuN(+) nuclei became gradually larger than that of the 7-AAD(+) nuclei, although the areas of the 7-AAD(+) and NeuN(+) nuclei correlated to a significant degree (<i>r</i> = 0.917, <i>P</i><0.001).</p

Asymmetry of the nuclei densities in the FPC.

<p>(A) 7-AAD(+), (B) NeuN(+), (C) olig2(+), and (D) NeuN(−)/olig2(−) nuclei. Left-right asymmetry rates (%) were calculated using the following formula: (the mean nuclei density in the left hemisphere)/(the mean nuclei density in the right hemisphere) ×100–100 (%). The positive % indicates left-greater-than-right asymmetry in the nuclei densities and the negative % vice versa. * <i>P</i><0.05, **<i>P</i><0.01, two-factor factorial ANOVA (diagnostic group×brain hemisphere).</p