Fig. 4

Endosomal alterations in TSPAN6-overexpressing cells. a EM images showing enlarged compartments in HEK293 cells overexpressing TSPAN6. HEK293 cells expressing an empty vector (control) present endosomes (a a) showing a normal aspect and multivesicular bodies (MVBs; a c and a e) containing normal-sized intraluminal vesicles (ILVs, empty arrowheads). In comparison, HEK293 cells overexpressing TSPAN6 show a higher number of endosomal compartments (a b), some of them with non-degraded material in the lumen (a b, arrowheads) and MVBs with enlarged ILVs (ad and a f; arrowheads). The nucleus is indicated with an N. b Quantification of the number of endosomes from TEM images of HEK293 cells like those shown in panel a. Overexpression of TSPAN6 increases the number of endosomal compartments per cell (n = 72 control cells; n = 59 TSPAN6-transfected cells; 2 independent experiments). c Analysis of the size of endosomal compartments from TEM images. Larger endosomal compartments are found in HEK293 cells overexpressing TSPAN6 in comparison to controls cells expressing an empty vector (n = 167 endosomes for control cells; n = 267 for TSPAN6-transfected cells; 2 independent experiments). d Determination of the surface of ILVs from TEM images like those shown in panel a. All the ILVs from the control or the TSPAN6-overexpressing cells were grouped according to their size in μm² (n = 202 ILVs from control cells; n = 204 ILVs from TSPAN6-overexpressing cells; 2 independent experiments). This demonstrates enrichment for larger structures in the TSPAN6 overexpressing cells. The mean of the surface plotted for each range shows larger ILVs in cells overexpressing TSPAN6 when comparing ILVs larger than 0.1 μm². Statistical significance was determined by t- test (*p <0.05, ***p <0.001)