Fig 2: Analysis of AnxA2 function in G2019S primary striatal astrocytes at endogenous level. A Western blot analysis of primary striatal astrocyte lysates under PFF-treated and basal conditions using anti-Lrrk2, anti-a-syn, and anti-AnxA2 antibodies. B, C, D Quantification of band intensity was performed using ImageJ and normalized by ß-actin (n = 4). E Representative images of Lrrk2+/+ and Lrrk2GS/GS astrocytes treated or not with a-syn PFFs and stained with anti-AnxA2 (green), anti-a-syn (red), F-actin (cyano), and cell nuclei with DAPI (blue). MLi-2-treated Lrrk2GS/GS astrocytes are shown in the bottom panels. Scale bar 20 µm. Insets show a close-up of a-syn inclusions and re-localized AnxA2. F, G Eight images per cell culture were analyzed (n = 3). Quantifications of Anxa2 puncta and AnxA2-a-syn PFFs proximity were performed using ImageJ. Statistical analysis in F and G was made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis in B, C, and D was performed with unpaired t test or one-way ANOVA followed by Tukey’s multiple comparisons test. *p = 0.05, **p = 0.01, ***p = 0.001

Fig 3: Investigation of AnxA2 function in astrocyte-mediated a-syn phagocytic clearance. A Unlabeled a-syn PFFs have been applied to primary astrocytes for 24 h. Projections verify the proximity of the internalized a-syn fibrils and endogenous AnxA2. Cell cytoskeleton (F-actin, cyano), a-syn (red), AnxA2 (green), DAPI (blue). Scale bar 20 µm. B Schematic outline of the experimental setup. Cells were transfected using 3xFlag-GFP-encoding plasmid together with scramble or AnxA2 siRNA. 48 h post transfection cells were treated with 0.5 µM sonicated a-syn PFFs for 24 h and imaged using confocal microscopy. C Representative images of primary striatal astrocytes transfected with scramble or Anxa2 siRNA together with a GFP-encoding plasmid in a-syn PFF-treated condition. Projections verify the proximity of the internalized a-syn fibrils; GFP (green), a-syn (red), DAPI (blue). Scale bar 30 µm. D Four images per cell culture were analyzed (n = 3). Quantifications of a-syn PFFs fluorescent-positive puncta were performed using ImageJ (ComDet plug-in). E Western blot analysis of primary striatal astrocyte lysates transfected with scramble and AnxA2 siRNA under basal and PFF-treated conditions. Anti-Lrrk2, anti-a-syn, and anti-AnxA2 antibodies have been employed. F, G, H Quantification of band intensity was performed using ImageJ and normalized by ß-actin (n = 3). Statistical analysis in D was made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis in F, G, and H was performed with an unpaired t test or one-way ANOVA followed by Tukey’s multiple comparisons test. *p = 0.05, **p = 0.01, ***p = 0.001