Fig. 5

Effect of CD63 knockdown on exosome secretion and endosomal pathology in DS cells. 2N and DS fibroblasts were transfected with either CD63 or negative control siRNAs. a CD63 knockdown was confirmed by Western-blot analysis of cell lysates. b Over 3 days, exosomes were collected from the cell culture media and quantified by Western-blot analysis for the exosomal markers CD63, TSG101, and Alix. c No significant changes were observed in exosome release by 2N cells following CD63 silencing compared to controls. d DS fibroblasts in which CD63 was silenced showed decreased release of exosomes as seen by lower levels of exosomal TSG101 and Alix as compared to control DS cells. Student t-test, n = 4 independent experiments (*p < 0.05; ***p < 0.001). e Early endosomes were immunolabeled with an anti-EEA1 antibody of transfected 2N and DS cells (calibration bar = 20 μm). f No significant changes in the endosomal number were detected in CD63-reduced 2N fibroblasts compared to control 2N cells, while a significant increase in number of endosomes was observed in DS cells following CD63 knockdown. g No significant differences were found in the area occupied by endosomes in 2N and DS cells after knocking down CD63, however DS fibroblasts showed a trend for an increase. Note that the number and area occupied by endosomes in DS fibroblasts is significantly higher than in 2N under basal (control-siRNA) conditions (f, g). Area is expressed in pixels per cell. One-way ANOVA followed by Tukey post-hoc multiple comparison test, n = 4 independent experiments (**p < 0.01; ***p < 0.001; ****p< 0.0001)