IST1 structures surround markers of extracellular vesicles and are left behind by the cells. (A) moDCs immunolabeled for IST1 (magenta in merge) and CD63, CD9, LAMP1, or ubiquitin (green). Blue: DAPI. Line graphs show fluorescence intensity profiles as indicated by the yellow line. Yellow shaded areas show regions where the fluorescence intensities for both proteins exceed the 50% intensity. (B) TIRF microscopy of moDCs co-expressing GPI-anchored RFP (green) with GFP-labeled IST1 (magenta; n = 3 donors). Scale bar: 5 μm. (C) Confocal micrographs of moDCs cultured for the indicated times and immunolabeled for IST1 (magenta) and phalloidin (green). Yellow arrowheads: IST1 structures inside (15 min) and outside (2 and 24 h) the cell. Left graph: average number of IST1 structures per cell localized inside or outside the cell (as determined by phalloidin; n = 4 donors). Only structures with a surface area >0.3 μm2 were included in this analysis. Right graph: average size of the structures. Time points are compared to the first time point (15 min) using a two-way ANOVA followed by Dunnet’s post hoc test. Data distribution was assumed to be normal, but this was not formally tested. **: P < 0.01; ****: P < 0.0001; NS: not significant. (D) Top: Confocal micrographs of moDCs immunolabeled for IST1 (magenta), HLA-DR (cyan), phalloidin (yellow), and DAPI (blue). Bottom: Same staining of patches of left behind IST1-positive structures, including orthogonal views. Yellow lines indicate where the cross-section was taken. Scale bars: 10 μm unless indicated otherwise.
