Fig. 8.

Internalization and endocytic trafficking of sEVs in primary astrocytes upon methyl-β-cyclodextrin treatment. Cells, treated with Dil-labeled sEVs (red), in the absence (NT) or presence of methyl-β-cyclodextrin (Cyclo) for 6 h and 24 h, were fixed and immunostained against α-Tubulin (α-Tub) (gray) and DAPI (blue). Representative Imaris images show the internalization of sEVs and in/mem sEVs without or with methyl-β-cyclodextrin 6 h and 24 h (a) post-treatment. ‘’sEVs mem’’ were depicted with arrowheads. Scale bar 10 μm. Graphs present the total volume of internalized sEVs per cell (b), the number of sEV puncta per cell (c), the mean volume of internalized sEVs (d), the percentage of sEV volume, inside (e) or membrane (f) per total volume, 6 h and 24 h post-incubation. Colocalization of sEVs with Rab5 (g–i) and Lamp1 (j–l) was monitored. Scale bar 5 μm. Graphs show colocalization between sEVs and Rab5 (h) or Lamp1 (k) and the mean volume of colocalized puncta (i and l) at different time points, with or without methyl-β-cyclodextrin. Data are presented as the mean ± SEM of minimum three independent cell preparations, with at least two replicates per assay; one-way ANOVA with Tukey’s correction was used for (d), (i) and (l), two-way ANOVA with Tukey’s correction for (b), (e), (f), (h), and (k) and multiple t test for (c). Statistical significance was set as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001