Fig. 1

ASC specks formation upon PFFs stimulation correlated with progression of α-synuclein pathology. a Representative immunofluorescence staining of ASC (green) in BV2 cells stimulated with 1ug/ml LPS for 4 h followed by 5 mM ATP for 0.5 h or 2 μM human A53T mutant α-synuclein PFFs for 16 h. ATP was used as the positive control. DAPI represents the nuclear signal (blue). The white dotted boxes in images are magnified on the right. White arrows indicate ASC specks. b Quantification of the percentages of ASC specks-positive BV2 cells. c–f Immunoblot (IB) analysis and quantification of phosphate-α-synuclein aggregates and ASC levels in bilateral striatum of controls and human A53T mutant α-synuclein PFFs-induced PD mice at six- and twelve-weeks post-surgery (n = 3). The red dotted boxes in images indicate monomeric, dimeric or trimeric proteins. g A simple linear regression was performed on relative levels of ASC and phosphate-α-synuclein aggregates, and R squared was calculated (n = 9). h Representative immunofluorescence images of ASC (green), NLRP3 (red) and Iba1 (grey) in the ipsilateral striatum of indicated mice brains (n = 3). DAPI represents the nuclear signal (blue). The white dotted boxes in images are magnified on the right. White arrows indicate ASC specks. i Quantification of relative fluorescent intensities of NLRP3. (j) Quantification of relative fluorescent intensities of ASC. k Quantification of the average numbers of ASC specks per microglia in the indicated mice ipsilateral striatum. Data are presented as mean ± SEM for at least n = 3 and are analyzed by one-way or two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Significance levels are indicated as: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ^#p < 0.05; ^###p < 0.001. Scale bars are as indicated. L left, R right, LPS lipopolysaccharide, ATP adenosine triphosphate