Fig. 6.

Internalization and endocytic trafficking of sEVs in primary astrocytes following Dynasore treatment. Cells, incubated with Dil-labeled sEVs (red), in the absence (NT) or presence of Dynasore (Dyn) for 6 h and 24 h, were fixed and immunostained against α-Tubulin (α-Tub) (gray) and DAPI (blue). Representative Imaris images depict the internalization of sEVs masked with the α-Tub surface and in/mem sEVs in cells treated without or with Dyn at 6 h and 24 h (a). ‘’sEVs mem’’ were depicted with arrowheads. Scale bar 10 μm. Graphs show 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, after 6 h and 24 h of treatment. The endocytic trafficking of sEVs was monitored by measuring the colocalization of sEVs with Rab5 (g–i) and Lamp1 (j–l). Scale bar 5 μm. Graphs show the colocalization between sEVs and Rab5 (h) or Lamp1 (k) and the mean volume of colocalized puncta (i and l, respectively) at different time points. 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 < .01, ***p < 0.001, ****p < .0001