Fig. 3

EV-delivered ROR1/2 mainly accumulates at the extracellular site of the target cell plasma membrane after 24 h. A Immunofluorescence: MCF-7 cells were stimulated for 24 h with 10 µg/ml lEVs or sEVs from empty vector control (pCMV/pcDNA) or ROR1/2 overexpressing (pROR1/pROR2) cells and ROR signals were visualized by confocal microscopy. Scale bar: 10 µm. B Confocal microscopy: Immunofluorescence-based co-localization of ROR1/2 with EpCAM, EEA1 or LAMP2 in MCF-7 cells stimulated for 24 h with lEVs (upper panel) or sEVs (lower panel) isolated from MCF-7 pROR1 or pROR2 cells. Scale bar: 10 µm. C MCF-7 cells were stimulated with EVs from pROR2 cells for 24 h, then treated with/without trypsin for 90 s at 37 °C (n = 3). Boxplots depict the median (line), the 25–75 percentiles (box) and the 10–90 percentiles (whiskers) of n = 15 quantified fields. Scale bar: 10 µm