4506 IRX1 inﬂuences peritoneal spreading and metastasis J Jiang et al Cell adhesion assay Adhesion of tumor cells to ECM is a pivotal step in developing tumor dissemination. The CytoSelect 48-well cell adhesion kits (ﬁbronectin-coated and collagen IV-coated; Cell Biolabs, Inc., San Diego, CA, USA) were used in this experiment. The microtiter plates were warmed up at room temperature for 10 min. The RPMI1640 was prepared according to instruction contained 0.5% bovine serum albumin, 2 mM CaCl2 and 2 mM MgCl2, and used to resuspend the cells of SGC-7901, SGC- 7901/vector and SGC-7901/IRX1. A 150 ml of 1 106 cells/ml suspension was added to each well and incubated for 60 min. Non-speciﬁc protein binding of the coated wells was blocked by adding 1% (w/v) bovine serum albumin (41C, 12 h) and rinsing the wells with Hepes buffer. Non-adherent cells were removed by washing three times with PBS. A 200-ml cell stain solution was added to each well and retained for 10 min at room temperature, discarding stain solution and washing three times with PBS. The total adhesive cells were determined by dissolving the attached cells in the microtiter wells with 200 ml cell extraction solution. The absorbance of each well was measured at 560 nm with microtiter plate reader (TD-20/20 analytical luminometer, Sunnyvale, CA, USA). OD showed a linear relationship to the amount of cells and was corrected for dying of bovine serum albumin/polystyrene without cells. Chick embryo CAM assay Fertilized chicken eggs (Xing-Huo farm, Shanghai, China) were incubated at 37 1C with humidity of 70%. On the 6th day of incubation, a round window was opened on the shell. Normally developed embryos were used for experiment. Malformated or dead embryos were excluded from experi- ment. The open window was sealed with transparent tape and incubated overnight. On the 7th day, a piece of sterilized ﬁlter paper disc (0.5 cm diameter) was placed on the surface of the CAM. A 30-ml collected supernatant from SGC-7901, SGC-7901/vector and SCG-7901/IRX1 was dropped to ﬁlter paper disc daily and sealed with transparent tape for consecutive 3 days. On the 10th day of incubation, the eggs were photographed with MacroPATH dissecting microscope (Milestone, Italy). The vessel numbers around the ﬁlter paper disc were assessed. All vessels vertically to the disc or intersected with the disc at an angle greater than 451 were counted. Nude mice study In this experiment, both peritoneal spreading and pulmonary metastasis were observed. Four-week-old male BALB/C nude mice were purchased from the Institute of Zoology, Chinese Academy of Sciences of Shanghai. All experiments were performed in accordance with the ofﬁcial recommendations of the Chinese animal community. In peritoneal spreading study, 10 mice were enrolled in each group. In all, 2 107 cells/ ml of SGC-7901, SGC-7901/vector and SCG-7901/IRX1 were resuspended. A 0.1-ml suspension per mouse was injected intraperitoneally. In pulmonary metastasis study, 15 mice were enrolled in each group. In all, 2 107 cells/ml of SGC-7901, SGC-7901/vector and SCG-7901/IRX1 were resuspended. A 0.1-ml suspension per mouse was injected into caudal vena. On the 30th day after intraperitoneal injection, mice were killed by cervical decapitation. The abdominal masses were excised and ﬁxed with 10% buffered formalin for further morphological analysis. On the 60th day after caudal vena injection, mice were killed by cervical decapitation. The thoracic cavity of mice was examined carefully. The pulmo- nary metastatic masses were removed and ﬁxed with 10% buffered formalin for further morphological analysis. Oncogene Histochemistry and immunohistochemistry All parafﬁn-embedded specimens were cut into serial 5 mm sections. The sections were deparafﬁnized, rehydrated and subjected to histochemical and immunohistochemical staining. The immuno- histochemistry was conducted with monoclonal rat anti-mouse CD34 (1:50 dilution; Abcam, Cambridge, MA, USA), mouse anti-human BDKRB2 (1:50 dilution; Abcam), rabbit anti-human IRX1 (1:50 dilution; Bioworld, Louis Park, MN, USA). Dako two-step detection Kit was used (Dako, Carpinteria, CA, USA). The histochemistry was performed with PAS staining. The slides were counterstained with hematoxylin and viewed with light microscope. Microvessel density and vascular mimicry density per mm2 were calculated, respectively, in ﬁve random ﬁelds. Chromatin immunoprecipitation Chromatin immunoprecipitation assays were performed accord- ing to the kit manual (Upstate, Billerica, MA, USA). SGC-7901 cells were cultured in 1640 culture medium with 10% fetal serum, till the density of 90%, and then collected and subjected to sodium dodecyl sulfate lysation after formaldehyde cross- link of DNA and proteins at room temperature for 10 min. After sonication, antibodies of normal mouse IgG and speciﬁc antibodies to IRX1 (Abcam) were added and mixed overnight with rotation. After adding the protein G agrose to collect the antibody/antigen/DNA complex, the complex then undergo washing and elution, and reverse crosslinked by 5 M NaCl treat- ment in 65 1C for 5 h. Puriﬁed chromatins were then analyzed by PCR with speciﬁc primers for BDKRB2 gene, along with control primers to detect GAPDH gene. RNAi experiments To verify the association of candidate genes with the biological functions, RNAi technology was used. SGC-7901 cells (3 105 cells) were plated onto six-well plates until 50% conﬂuence. The cells were transfected with siRNA (GenePharma Co., Shanghai, China) targeting BDKRB2, IRX1 and PAK1 in accordance with the manufacturer’s instructions (the sequences of siRNAs were listed in Supplementary Table). Brieﬂy, a 10-ml transfection reagent was incubated with 500 ml OPTI-MEM medium (Gibco, Carlsbad, CA, USA) for 5 min. Subsequently, siRNAs were added to wells (120 nmol/l). After 2 days trans- fection, cells were collected for protein expressing analysis. Invasion, migration and cell viability assays Cell invasion and cell migration assay were performed. The transwell chamber was used (8 mm, 24-well format; Corning, Lowell, MA, USA). In invasion assay, the insert membranes were coated with diluted Matrigel (BD Biosciences). Cells (1 105) were added to upper chamber and cultured for 48 h. In migration assay, the insert membranes were not coated with Matrigel and cultured in the same condition. Finally, the insert membranes were cut and stained with Crystal violet (0.04% in water; 100 ml) and counted the permeating cells under the inverted microscope and photographed. Meanwhile, the cell viability (1 105) of different groups with same culture conditions was also assayed at 0, 24 and 48 h time points by cell counter (Coulter, Sequim, WA, USA). At least three independent experiments were performed for all conditions. The data are shown as means±s.d. Protein analysis Protein lysates were prepared from collected cells in lysis buffer containing 20 mM/l Hepes, 0.15 M NaCl and 1% Triton X-100 supplemented with 80 ml/ml phosphatase inhi- bitor cocktail II (Sigma, St Louis, MO, USA) and 10 ml/ml