Fig. 4.

sEVs are targeted to the endocytic pathway and are colocalized with Rab5 (EE) and Lamp1 (LE/Lysosomes) following addition on primary astrocytes. Cells were incubated with Dil-stained sEVs (red) for 6 h and 24 h, and colocalization of sEVs with Rab5 and Lamp1 was measured 6 h and 24 h post-treatment. Cells were fixed and immunostained for Rab5 and Lamp1 (green), α-Tubulin (α-Tub) (gray), and DAPI (blue). Representative Imaris images depict colocalization between internalized sEVs and the endocytic markers, Rab5 (a–c) and Lamp1 (d–f), after 6 h and 24 h of treatment. The colocalization surface is depicted in yellow. Scale bar 5 μm. Graphs show the colocalization between sEVs and Rab5/Lamp1 (Manders’ colocalization coefficient) after 6 h and 24 h (b and e) of treatment as well as the mean volume of puncta colocalized with Rab5 (c) and Lamp1 (f). Data are presented as the mean ± SEM of minimum three independent cell preparations, with more than 60 cells measured; Student's t test was used, and statistical significance was set as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001