Fig. 10.

Uptake of sEVs in primary astrocytes through the actin-dependent endocytic pathway. Cells, incubated with Dil-labeled sEVs (red), in the absence or presence of Cytochalasin D (Cyto) for 6 h and 24 h, were fixed and immunolabeled against α-Tubulin (α-Tub) (gray) and nuclei were stained with DAPI (blue). Representative Imaris images show the internalization of sEVs and in/mem sEVs without or with Cyto, 6 h and 24 h (a) post-treatment. ‘’sEVs mem’’ was 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 evaluated. 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, respectively) at different time points, with or without Cyto. 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 (e), (f), (h), and (k) and multiple t test for (b) and (c). Statistical significance was set as *p < 0.05, **p < 0.01, ***p < .001, ****p < 0.0001