Dll4 is transferred to other cells via exosomes. (A) U87GM cells were seeded onto 6-well plates at 3 × 105/well. The cells were then incubated with either control exosomes (50 μg/mL) or Dll4-containing exosomes (50 μg/mL) in Opti-Mem for 24 hours before extensive washing with PBS and lysis in radioimmunoprecipitation assay buffer. Lysates were subjected to Western blotting and were probed with either anti-Dll4 or β-tubulin as a loading control. Exo indicates exosomes. (B) The same experiment was repeated, except the cells were fixed in acetone after the 24-hour incubation. They were then stained with anti-hDll4. Images were taken on a Nikon Eclipse E800 microscope at 40× magnification. (C) U87 cells incubated with exosomes (50 μg/mL) for 24 hours were pelleted, embedded, and sectioned. The sections were then stained with anti-hDll4, and images were taken at 50× magnification with a Nikon Eclipse E800 microscope.
