Dll4-containing exosomes inhibit Notch signaling. (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 harvested from U87 (50 μg/mL). After 24 hours, the cells were extracted in TRI reagent to isolate the RNA. Quantitative polymerase chain reaction protocol was conducted to examine the Notch target genes Hey1 and Hey2. (B) Identical experiment with exosomes isolated from HUVEC control cells or HUVECs plated on Dll4. 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. (C) The same experiment as shown in panel A, except after 24 hours, the cells were lysed in radioimmunoprecipitation assay buffer and subjected to Western blotting to detect the level of NICD or β-actin as a loading control. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase; and exo, exosomes.
