Dll4-containing exosomes reduce the level of stalk cell markers and confer a tip cell phenotype. (A) HUVECs were plated onto bovine serum albumin (BSA) or Dll4-coated 6-well plates at a density of 3 × 105/well in the presence of either control exosomes (50 μg/mL) or Dll4-containing exosomes (50 μg/mL). After 24 hours, the cells were extracted in TRI reagent to isolate the RNA. Quantitative polymerase chain reaction protocol was performed to examine the stalk cell markers Jagged-1 and vascular endothelial growth factor receptor-1 (VEGFR1). Samples were analyzed in triplicate. One-way analysis of variance with a Tukey multiple-comparison post hoc test was performed with GraphPad Prism 4.0b software. Exo indicates exosomes. (B) HUVECs were incubated with 3 different batches of control or Dll4-containing exosomes for 24 hours before staining with tetramethylrhodamine B isothiocyanate–phalloidin to visualize the filopodia. (C) The filopodia were counted on 40 cells from each condition, and the length of the longest 3 filopodia on each cell was assessed with ImageJ. Images were taken on a Zeiss Axioskop 2 microscope at 63× magnification. Data were analyzed by t test with GraphPad Prism 4.0b software and are shown as mean with SD.
