Fig. 5
TSPAN6 is found in ILVs and enhances the formation of exosomes. a Representative staining of 2 independent experiments showing HEK293 cells overexpressing the Rab5Q79L mutant together with an empty vector (upper panels) or myc-TSPAN6 (lower panels). Immunofluorescence studies were carried out with monoclonal antibodies against myc (to detect TSPAN6), APP-CTF and Rab5. APP-CTF is observed in the lumen (filled arrowheads) and the limiting membrane (empty arrowheads) of endosomes in both conditions; however, cells overexpressing myc-TSPAN6 show enlarged endosomes. Scale bar = 10 μm. b Percentage of APP-CTF in the lumen or the rim of endosomes in cells overexpressing an empty vector (n = 109 endosomes; 2 independent experiments) or myc-TSPAN6 (n = 106 endosomes; 2 independent experiments). c The average area (pixels2) of Rab5Q79L endosomes in cells overexpressing myc-TSPAN6 (n = 75 endosomes; 2 independent experiments) was increased in comparison to control cells expressing an empty vector (n = 65 endosomes; 2 independent experiments). d Western blot analysis of total HEK293 lysates (transfected with Flag-TSPAN6 or left untransfected) or the exosomal fraction (Exo) isolated from their conditioned medium. The blot was probed with a monoclonal antibody against FlagM2 and a polyclonal antibody against TSPAN6 to assess the overexpression of protein, a monoclonal antibody against calnexin (non-exosomal protein), a polyclonal antibody against Tsg101 (exosomal protein) and a monoclonal antibody against actin. e The western blot of total HEK293 lysates and their exosomal fractions shows no differences in the APP-CTF levels for the same amount (2.5 μg) of exosome lysates when TSPAN6 is overexpressed. f Quantification of the concentration of secreted exosomes (vesicles/ml) by the nanovesicles tracking system Nanosight from control HEK293 cells or overexpressing TSPAN6 (3 independent experiments per condition), shows that TSPAN6 increases the secretion of exosomes. g Graph showing the distribution of the concentrations of the vesicles grouped by size (nm) and found in the exosomal fractions of TSPAN6-overexpressing HEK293 cells and control cells (data from panel f). TSPAN6-overexpressing cells show a higher concentration for all the vesicle sizes. Statistical significance was determined by t-test (**p <0.01)