Breast Cancer Res Treat (2012) 134:943–955 BrdU incorporation assay DNA synthesis was determined by BrdU labeling assay using Cell Proliferation ELISA, BrdU (colorimetric) kit. In brief, 3,000 HUVECs per well were seeded in a gelatin- coated for overnight attachment. Then the cultivated medium was replaced with serum-free medium supple- mented with 20 ng/ml VEGF as well as different concen- trations of ellagic acid in a ﬁnal volume of 100 ll/well. After 24 h, cells were labeled with BrdU (2 h, 37 C), incubated with FixDenat solution (30 min, 20 C), and re- incubated with Anti-BrdU POD (90 min, 20 C). The sample absorbance was ﬁnally detected in an ELISA reader at 450 nm. Lactate dehydrogenase (LDH) toxicity assay The LDH released into cell cultures is an index of cyto- toxicity and evaluation of the permeability of cell mem- brane. HUVEC were seeded in 96-well plate at a density of 3,000 cells per well. After incubation with various con- centrations of ellagic acid for 24 h, cell supernatants were collected and analyzed for LDH activity using LDH cyto- toxicity assay kit from Caymen Company. The absorbance of formed formazan was read at 490 nm on a microplate reader. Wound-healing assay HUVEC cells were seeded in a 6-well plate at a density of 5 9 105/well. After the cells had reached 90% conﬂuency, a scrape was made in the middle of the plate by a 10 ll tip with the gap widths between 150 and 200 lm. After scrapping, the cells were washed with PBS twice, and then incubated with fresh medium containing VEGF with or without ellagic acid (10 lM). Migration ability was eval- uated by measuring the gap widths narrowed down by HUVECs movement at different time intervals of 0, 12, and 24 h. Images were then taken with a microscope video system, and three places of each gap were measured and averaged. Invasive assay Transwell model (8-lm pore size) was applied to perform the invasive assay. The upper side of transwell was coated with Matrigel, while the lower compartment was coated with type I Collagen. Then the pre-coated transwell was placed into a well of 6-well plate containing 1 ml complete M199 medium. HUVECs (2 9 105 cells per well) were suspended in 1 ml medium containing various concentra- tions of ellagic acid and VEGF, and then added to the 945 upper chamber. After incubation at 37 C for 24 h, the non- migrated cells in the upper surface of membrane were removed with a PBS-soaked cotton swab. Then the mem- branes were ﬁxed with 4% PFA for 30 min. Cells that migrated to the lower side of the membranes were stained with HE method. Photographs were taken by a microscope video system. The number of cells that crossed the mem- brane in the treated group was compared with that in the control group. Tube formation assay The tube formation assay was performed using 12-well plate coated with 100 ll Matrigel basement membrane matrix (BD Bioscience, Bedford, MA) per well and poly- merized at 37 for 30 min. HUVECs suspended in M199 medium containing 2% FBS were plated on the Matrigel at a density of 2 9 105 cells/well. Different concentrations of ellagic acid (2.5, 5, and 10 lM) were then added together with VEGF (20 ng/ml). After 8 h, The Matrigel-induced morphological changes of HUVECs and their formed tube networks were observed under a microscope and photo- graphed at a ﬁve-fold magniﬁcation. Kinase activity detection In vitro VEGFR-2 tyrosine kinase activity was assayed using an enzyme-linked immunosorbent assay kit (Boeh- ringer Mannheim, SA). In brief, ellagic acid was incubated with VEGFR-2 (Upstate) in assay buffer containing Mg2? and ATP in 96-well plate coated with a poly-Glu-Tyr substrate. Phosphorylated tyrosine was then detected by sequential incubation with a mouse IgG anti-phosphoty- rosine antibody and an HRP-linked sheep anti-mouse immunoglobulin antibody. Color was developed with an HRP chromogenic substrate and quantiﬁed by an ELISA reader at wavelength 450 nm. The results were expressed as percent kinase activity. Western blotting In brief, cell lysates (30 lg) were separated by 8% SDS- PAGE and transferred to polyvinylidene diﬂuoride mem- branes. Membranes were then incubated with primary antibodies including phosphorylated and/or total VEGFR- 2, ERK, AKT, JNK, eNOS, and b-actin (Cell Signaling Technology, Danvers, MA). After overnight incubation at 4 C, membranes were washed with TBST three times and then incubated with secondary antibodies at room tem- perature for 2 h. Immunoreactive bands were then visual- ized by the enhanced chemiluminescence (ECL) detection system (GE healthcare). 123