The products were transformed into DH5α competent cells. Ampicillin-resistant colonies were chosen, identified by restriction digestion and further confirmed by DNA sequencing. SGC7901 cells were planted in six-well plates and CHIR-99021 mw cultured in drug-free medium. At 90-95% confluence, cells were washed twice with PBS, grew in 2 ml of DMEM without antibiotics. Using Lipofectamine™ 2000 reagent (Invitrogen, Inc. Carlsbad CA), 2 μg of mU6pro-COX-2siRNA plasmids were transfected into cells according to the manufacturer’s instructions. The cells transfected with mU6pro vector alone were served as negative control. Forty-eight hours later, cells were placed in growth medium containing G418

(GIBCO) for clone selection. The expression see more levels of COX-2 in G418-resistant clones were evaluated by western blot analysis. RT-PCR All of the PCR products were separated on ethidium bromide stained agarose, and visualized with UV as described previously [6]. Western blot analysis The western blot was done as described previously. In brief, total cellular proteins were prepared and then quantified by Bradford method [7]. A Dinaciclib measure of 80 ug of lysates were electrophoresed in 12% SDS-PAGE and blotted

on a nitrocellulose membrane (Immoblin-P, Millipore, Bedford, MA, USA). Membranes were blocked with 5% fat-free milk powder at room temperature and incubated overnight with antibody at 4°C. After three washes for 15 min in PBS-T, the membrane was incubated with the HRP-conjugated goat anti-mouse IgG antibody (Wuhan, Hubei, China) for 1 h at room temperature. The enhanced chemiluminescence (Amersham Life Science, Piscataway, NJ, USA) was added and monitored for the development of color. Cell growth assay Cells were seeded on a 96-well plate at 3 × 104 cells/well. Each sample had four replicates. The medium was replaced at 2-day intervals. Viable cells were counted by the 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide (MTT) assay after 2, 4, 6, and 8 days. Tumor growth in nude mice Female athymic nu/nu mice, 5-6 weeks of age, were obtained

from FMMU Experimental Animal Co. (Shaanxi, China) and housed in a pathogen-free facility for all of the experiments. The logarithmically growing cells were trypsinized and resuspended PLEKHB2 in D’Hanks solution, and 5 × 106 cells in 0.2 ml were injected subcutaneously into the left flank of mice [8]. Experimental and control groups had at least 6 mice each. Tumors were measured twice weekly with microcalipers, and the tumor volume was calculated according to the formula: volume = length × (width2)/2. Quantification of tumor microvessel density Tumor microvessel densities (MVD) were quantified by anti-CD31 immunohistochemistry. Briefly, tumor sections from nude mice were cut using a LEICA cryostat and the paraffin sections were mounted on positively charged Superfrost slides and dried overnight. The immunostaining was done according to standardized protocols.