Figure 2

Effect of 6-ME on VEGF-induced survival, migration of endothelial cells and tube formation in vitro and phosphorylation of Akt and p38 MAPK. (A) HUVE cells were serum starved in 5% FBS M199 supplemented with heparin and pen/strep. Then cells were stimulated or not with VEGF (50 ng/ml) in the presence or absence of 6-ME (10μM) for 18 h. Floating and adherent cells were analyzed by flow cytometry. The number indicates the percentage of the hypodiploid cells. The experiment shown is a representative one from three independent experiments. (B) Confluent HUVE cell monolayers were wounded with a sterile tip prior to serum-starvation in M199 supplemented with 5% FBS, heparin and pen/strep. Then, cells were induced by VEGF (50 ng/ml) in the presence or absence of 6-ME (10μM) and placed in a 37°C, 5% CO₂ chamber and monitored using a Leica DM IBRE microscope equipped with a HRD060-NIK CCD-camera and metamorph software. The graph shows images taken from non-induced cells at time points 0 and 600 min (at 600 min we have the possibility of contribution from proliferation – I would show an earlier time point) and from induced cells at the same time points, in the presence or absence of 6-ME, representing the number of the cells per centimeter of wound ± s.d. derived from three independent experiments. (C) HUVE cells were seeded on polymerized matrigel in M199 supplemented with 5% FBS, 1% pen/strep and heparin at density 8 x 10⁴ cell/ml. Then cells were treated with DMSO or 6-ME at various concentrations for 12 h and tube formation was observed using an inverted microscope. (D) HUVE cells were serum starved for 2 h in M199 and then stimulated with VEGF (50 ng/ml), in the absence or presence of 6-ME, for 15 min. Then cell lysates were collected with 1% SDS lysis buffer, supplemented with PMSF, and immunoblotting followed using antibodies against endogenous phospho-Akt, actin, phospho-p38 and p38. Experiments shown in (A) and (B) are representative from three independent experiments.