Dll4-containing exosomes transfer Dll4 to the membrane, reduce Notch1 receptor levels, and are endocytosed. (A) Dll4-containing exosomes were labeled with a red fluorescent dye (PKH67) as described in “Methods.” HUVECs were incubated with Dll4-containing exosomes (50 μg/mL) for 16 hours before images were acquired with a Zeiss LSM 510 confocal microscope at 63× magnification. Images shown are a midsection slice through the cells showing intracellular localization of the exosomes and exclusion from the nucleus. Phase-contrast image and merged image show that fluorescence did not occur at the membrane. (B) HUVECs or U87GM cells were seeded at a density of 3 × 106 cells/150 cm2 dish. Each dish was incubated for 24 hours with control exosomes (50 μg/mL) or Dll4-containing exosomes (50 μg/mL) before the cell-surface proteins were biotinylated and purified as described in “Methods.” The purified proteins were subjected to Western blotting to detect Dll4 or Notch 1 at the cell membrane, with CD31 probed as a loading control. (C) The same experiment was repeated, except the cells were lysed to isolate total protein. The lysates were subjected to Western blotting to detect total Notch1, with β-actin used as a loading control. Exo indicates exosomes.
