Figure 3.

Compact packaging and rapid internalization of AREG exosomes. (A) Exosomes were isolated from conditioned medium of MDCK cells stably expressing EGFP-tagged AREG. The number of GFP molecules per exosome was determined as described in Experimental Procedures. Each exosome contains approximately 24 AREG molecules. (B) DiD-stained exosomes were purified from wild-type AREG-expressing MDCK cells and incubated with LM2-4175 cells for the indicated times. Flow cytometric analysis was performed as described in Experimental Procedures. Data represent the mean +/− SD. p < 0.005 (**). (C) Exosomes from non-tagged AREG-expressing MDCK cells were incubated with the fluorescent membrane stain DiD. LM2-4175 recipient cells were incubated with DiD-labeled AREG exosomes for the indicated times and uptake visualized by confocal microscopy. Scale bars represent 5 μm. (D) Exosomes from C-terminally EGFP-tagged AREG-expressing MDCK cells were purified and incubated with LM2-4175 cells for 30 min and internalization monitored by GFP fluorescence using confocal microscopy. Two xz planes are shown: apical surface (upper) and mid-cell (lower). Scale bars represent 5 μm. (E) LM2-4175 cells were pretreated in the presence (EGFR mAb) or absence (CTL) of 20 μg/mL EGFR mAb 528 for one hr and then incubated with DiD-stained AREG exosomes for 10 min. Flow cytometric analysis was performed as described in Experimental Procedures. Data represent the mean +/− SD. p<0.0001 (*).