Additional file 1 of Immunoregulation of microglial polarization: an unrecognized physiological function of α-synuclein
Creators: name: Li, Na
name: Stewart, Tessandra
name: Sheng, Lifu
name: Shi, Min
name: Cilento, Eugene M.
name: Wu, Yufeng
name: Hong, Jau-Syong
name: Zhang, Jing
Publisher: name: figshare
Year: 2020
Subjects: subject: Biophysics
subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure s1 Identification of α-synuclein (α-Syn)monomer, oligomer and primary microglia purity. a α-Syn monomer and oligomer are identified by western blot. Besides monomer, with a molecular weight around 15kDa, oligomer includes dipolymer, tripolymer, tetramer, pentamer with molecular weight ranging from 30kDa to 75kDa. b Representative images of immunofluorescence for microglia (Iba-1, red) or astrocyte (GFAP, red), respectively. Nuclei are counterstained with DAPI. (Scale bar=100μm, n=5, each containing 10-15 fields). c Iba-1+ and GFAP+ cells are quantified by counting at 10-15 randomly selected fields under each condition (*** p&lt;0.001). d Forms of α-Syn incubated in media for 6h, 12h and 24h. e Forms of injected exogenous α-Syn in both WT and SNCA-KO mice. f and g Comparison of anti-inflammatory effect of endogenous α-Syn between WT mice and SNCA-KO mice. (Scale bar=10μm, n=3, each containing 10-15 fields). Figure s2 α-Syn induces microglia towards a pro-inflammatory phenotype. a α-Syn induces iNOS expression post 24h treatment in primary microglia (n=3). b Densitometric analysis of relative intensity of iNOS expression. c Densitometric analysis of relative intensity of ARG-1 expression. d Nitrate concentration in supernatant at 24h under different concentrations of α-Syn. e-h mRNA levels of ARG-1, CD206, iNOS and CD16/32 in BV2 microglia with 6h treatment at different concentrations. i-l mRNA levels of ARG-1, CD206, iNOS and CD16/32 in BV2 microglia with 12h treatment at different concentrations. m-p mRNA of ARG-1, CD206, iNOS and CD16/32 in BV2 post treatment by physiological concentration of α-Syn for 24h. q Effect of a series concentrations of α-Syn monomer ranging from 0.05nM to 100nM on expression of iNOS and ARG-1 in BV2 microglia. One-way ANOVA with Newman Keuls Multiple Comparison Test. * p&lt;0.05, ** p&lt;0.01*** p&lt;0.001. Bar graphs show mean + s.e.m. Fig. s3. α-Syn oligomer exerts pro-inflammatory effects on microglia in a dose-dependent manner. a Oligomeric α-Syn induces iNOS, but not ARG-1 expression in primary microglia in a dose-dependent manner. b Densitometric analyses of relative intensities of iNOS and ARG-1 expressions (n=3 independent experiments). c mRNA levels of iNOS and ARG-1 in primary microglia under treatment with oligomer at 5pg/ml and 400pg/ml in BV2 cells (n=3). d Immunofluorescent staining of iNOS (red) and ARG-1 (green) in BV2 cells under different treatments (Scale bar=20μm). e Quantification of the percentage of iNOS and ARG-1 positive BV2 cells (n=5 replications each containing 15-20 fields). One way ANOVA with Newman Keuls Multiple Comparison Test. Bar graphs show mean + s.e.m. * p&lt;0.05, ** p&lt;0.01, *** p&lt;0.001. Figure s4 Neither α-Syn monomer nor oligomer activated ERK. a Primary microglia are treated with physiological concentration of α-Syn or higher concentration (400pg/ml) of oligomer for 12h. b Co-localization of α-Syn (green) and ERK (red) in microglia of mouse brain (Scale bar=10μm) Figure s5 Monomeric α-Syn does not alter levels of M2 phenotype-related JAK3-STAT6 pathway, CREB or PPARα. Western blots were performed to determine whether monomeric α-syn altered several pathways, but no major differences were apparent. Figure s6 Quantification of microglia state related molecule expressions by densitometric analysis. a-g Relative intensity of ERK, NF-κB, p- NF-κB, IKKα/β, IKBα,p- IKBα,PPARγ post treatment by α-Syn for 12h. One-way ANOVA with Newman Keuls Multiple Comparison Test. * p&lt;0.05, ** p&lt;0.01*** p&lt;0.001. Bar graphs show mean + s.e.m. Figure s7 Co-injection of monomeric α-Syn attenuates microglial inflammation induced by MPTP. Quantitative analyses of protein expression intensities normalized to GAPDH. One-Way ANOVA with Newman Keuls Multiple Comparison Test. Bar graphs show mean + s.e.m.* p&lt;0.05