BMC Cancer 2007, 7:95 http://www.biomedcentral.com/1471-2407/7/95 (VEGF 27 cycles) for 45s, or 57°C(β-actin 25 cycles) for 30s; extension at 72°C for 30s. The PCR products were analyzed by electrophoresis in a 2% agarose gel. The rela- tive RNA amount was calculated by the Gene Genius gel imaging system (Syngene, UK). All experiments were car- ried out three times: each time the reading was taken in triplicate and the average and standard deviations were calculated. Immuofluorescence double staining and semi-quantitative confocal laser scanning analysis Cells were fixed with methanol for 10 minutes at -20°C. After permeated with 0.5% Triton X-100 in PBS for 10 minutes at room temperature, cells were blocked by 2% bovine serum albumin for 30 minutes at 37°C. Then cells were incubated with the primary antibodies overnight at 4°C. Primary antibodies were anti-NF-κB mouse mono- clonal antibody (Santa Cruz, CA, USA) used at a dilution of 1:100, and anti-VEGF rabbit polyclonal antibody (Santa CruZ, CA, USA) used at a dilution of 1:150. After washing with TBST, the cells were incubated with second- ary antibodies, diluted 1:80 in TBST. Secondary antibod- ies that were used were CY3-conjugated goat anti-rabbit IgG (Sigma, St. Louis, USA) and fluorescein isothiocy- anate (FITC)-conjugated goat anti-mouse IgG (Pierce, Rockford, USA). The cells were then washed three times with PBS. Immunofluorescence microscopy was per- formed using a Leica TCS-SP2-AOBS-MP confocal micro- scope (Leica Microsystem, Heidelberg, Germany). According to the recommendation of Nakayama [13], the rate of the nuclear localization of p65 was calculated by the following method: count positive nuclear staining NF- κB from total cells and then calculate the percentage. Each of the five cell slides was counted every ten randomly selected in high-power fields (× 200). The relative protein amount of VEGF was calculated according to the mean intensity of the fluorescence on five cell slides by Leica confocal software 2.61. Stable Transfection of ACCs Cells with IκBαM and Control vector The PCMV-IκBαM vector was provided by Professor Chiao (Anderson Cancer center, Huston, Texa, USA). The CMV-IκBαM vector has mutations (S32, 36A) of the NH2 terminus and a COOH-terminal PEST sequence, which specifically inhibits phosphorylation of IκBα. Then ACC- M and ACC-2 cells (1 × 106) were transfected using 15 ul of lipofectamine reagent (Invitrogen, Grand Island, NY, USA) and 4 ug of PCMV-IκBαM or control PCMV vector according to the manufactures instruction. Cells were selected with standard medium containing 800 ug/ml and 600 ug/mlG418 respectively. Fourteen days later, neo- resistant colonies were isolated by trypsinization and established as subcultures. The cells resistant G418 and stable express exogenous IκBαM were used for subsequent analyses. Endothelial cell migration assay Endothelial cell motility assay was carried out as described previously [14]. Briefly, a 6-well plate was coated with type-I collagen and incubated overnight at 37°C. UVECs were seeded into the coated wells at a den- sity of 2 × 106 cells/well and incubated for 24 h. Then scrape the monolayer cells to make a clear area with a nar- row tip and wash with serum-free medium. 1.0 ml CM from ACC-M and ACC-2 cells were added into the wells and incubated for 24 h, and the 1.0 ml serum free DMEM medium was used as the control. Then the cells were fixed and stained using Acridine Orange and photographed by fluorescence microscope (Leica Microsystem, Heidelberg, Germany). Tube formation assay 500 ul type I collagen gel solution (0.3%) containing 5 mg/ml of human fibronectin (Collaborative Research Inc. Lexingtonf, USA), 100 ul 10 × DMEM and 400 ul NaOH- Heppers buffer was mixed in an ice cold condition and pipetted into a 6-well plate and kept for 30 min at 37°C or gelatinization. UVECs were seeded into the layer of the gel at a density of 5 × 104 cells/well with 10% FBS medium. After 24 h, the medium was replaced by 0.8 ml CM from ACC-M and ACC-2 cells, which was added with 0.2 ml 10% FBS DMEM medium to reach the 2% FBS-CM final concentration. The 1.0 ml 2% FBS medium was used as the control [15]. After 3 days, the tubular structures organized and gradually elongated and formed networks by UVEC cells. Then the cells were fixed and stained using Acridine Orange and photographed by a fluorescence microscope (Leica Microsystem, Heidelberg, Germany). In vitro three-dimensional angiogenic assay model In vitro three-dimensional angiogenic assay model was then performed as described previously [2]. Briefly, the type I collagen gel solution mixture was put on a filter of Millicell-CM inserts and placed in 6-well plates. After gelatinization of the collagen solution, UVEC cells (1 × 105 cells) were seeded on the surface of the gels in the Mil- licell and cultured in 10% FBS. When the UVEC cells reached subconfluence about 24 h after seeding, the medium in the outer wells was replaced by 1.5 ml 2.0% FBS-CM and the 1.5 ml 2.0% FBS medium was used as the control. In the Millicell well, 0.5 ml of 2.0% FBS medium was added. Within a week of culture, UVEC cells started to sprout into the gels beneath the confluent monolayer. Then the gels were fixed and stained using Acridine Orange, and the sprouting structures were observed using a Leica LSM 410 confocal laser scanning microscope. Page 3 of 7 (page number not for citation purposes)