Ergosterol peroxide (EP) exerts anti-angiogenic activity in vitro. (A) U266 cells were treated with various concentrations of EP (0, 6.25, 12.5, 25 or 50 μM) for 24 h. Cytotoxic effect of EP was evaluated by MTT assay. (B) U266 cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h. Cell lysates were prepared and subjected to Western blotting for VEGF. (C and D) U266 cells were seeded at density of 1 × 106 cells/well onto 6-well plates and treated with 25 μM EP for 0, 4, 8, 16 or 24 h (C) and various concentrations of EP (0, 6, 12.5 or 25 μM) for 24 h (D). Levels of VEGF in the supernatants were measured by using a Quantikine VEGF ELISA kit. (E) For in vitro tube formation assay, HUVECs (3 × 104 cells/well) were seeded onto Matrigel coated 24-well plates and treated with VEGF (20 ng/ml) in the absence or presence of EP (0, 12.5 or 25 μM) for 6 h. Cells were fixed with 2% paraformaldehyde, stained with 2% crystal violet, and the number of tube was randomly counted in selected areas (left). Representative photographs of tube formation in cells treated with VEGF (20 ng/ml) in the absence or presence of 25 μM EP (right). Each experiment was repeated three times and all data were expressed as means ± S.D. **, p < 0.01 vs untreated control. (F) HUVECs were treated with VEGF (20 ng/ml) in the absence or presence of 25 μM EP. Western blotting was performed for phopho-STAT3 and STAT3.