Fig. 9

CREB activation induces angiogenesis in vitro, depending on the EZH2/TSP1 axis. a Serum starved PC3 cells were treated with 10 μM ISO with or without 5 μM EZH2 inhibitor GSK126 or EPZ6438. Conditioned media (CM) from the PC3 cells were then used to culture serum starved SVEC-40 endothelial cells seeded on growth factor reduced Matrigel for angiogenesis tube formation assay. TSP1 peptides were added to 10 μM of final concentration to the SVEC-40 cells in conditioned media from ISO-treated PC3 cells. The assays have been done at least twice with similar results. b Conditioned medium from ISO-treated PC3 cells with EZH2 knockdown can not induce angiogenesis tube formation of SVEC-40 endothelial cells. c Conditioned medium from NE1.3 cells treated with 5 μM EZH2 inhibitor EPZ6438 or GSK126 were used in SVEC-40 tube formation assay. d Conditioned medium from serum starved LNCaP and NE1.3 cells was used as attractants for migration of SVEC-40 cells in Boyden Chamber migration assay. Representative pictures from two experiments are shown. e Western blotting of TSP1 and NE markers in PC3 expressing scramble shRNA or two independent TSP1 shRNAs. f Endothelial SVEC-40 migration assay with attractants of conditioned medium from PC3-scramble or PC3-shTSP1 cells, either mock treated or treated with 10 μM PKA/CREB signaling inhibitors PRO and ICI for 48 h. Representative pictures from two experiments are shown. Scale bar = 100 µm