Fig. 4

Stimulation with EVs derived from ROR1/2-overexpressing cells increases tumor invasion dependent on RhoA. A + B Boyden chamber invasion assays of MCF-7 cells after stimulation with (A) EVs isolated from MCF-7 empty vector (pCMV/pcDNA) or ROR1/2-overexpressing (pROR1/pROR2) cells or (B) EVs from MDA-MB-231 CRISPR control (Cr-CTL) or ROR1 KO (Cr-ROR1) cells (mean ± SD, n = 3). C Boyden chamber invasion assays of MCF-7 cells transfected with siRNA against RhoA (10 nM) 24 h prior to stimulation with EVs isolated from MCF-7 empty vector (pCMV/pcDNA) or ROR1/2-overexpressing (pROR1/pROR2) cells (mean ± SD, n = 2). D Immunofluorescence: MCF-7 cells were pre-treated with/without dynasore (12.5 µM) for 2 h and uptake of PKH26-labeled EVs or dye-only (PKH-CTL) into the cells was visualized by confocal microscopy (n = 3). Signals were quantified with ImageJ. 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. E Boyden chamber invasion assay of MCF-7 stimulated with pROR1/pROR2 EVs after pre-treatment of the cells with/without dynasore (12.5 µM) for 2 h (mean ± SD, n = 3)