4500 IRX1 inﬂuences peritoneal spreading and metastasis J Jiang et al Figure 2 Assays of microvessel density (MVD) or vasculogenic mimicry density (VMD) on tumor tissues. (a) MVD of tumor tissues was assayed by CD34 immunohistochemistry. A signiﬁcant reduction of MVD in tumor tissues of SCG-7901/IRX1 group, compared with mock or controls (3.8±0.83 vs 7.4±1.51 or 6.8±1.30, *Po0.001, the bar indicates 20 mm ). (b) VMD of tumor tissues was assayed by PAS histochemistry. A signiﬁcant reduction of VMD in tumor tissues of SCG-7901/IRX1 group, compared with mock or controls (1.20±1.30 vs 4.4±1.14 or 4.4±1.34, *P ¼ 0.002, the bar indicates 20 mm). with 5% CO2, tubular numbers of each group were assessed under light microscope. The supernatant from SGC-7901/IRX1 revealed strong inhibiting effect on tubular formation of HUVECs whatever in tubular number, tubular length and tubular intersecting nods, compared with mock or control (Figure 3). To examine the reproducibility of the ﬁnding, we assayed tubular formation after IRX1 overexpression on NCI-N87 gastric cancer cells. We got a similar result as described in SGC-7901 cancer cells. The result is presented in Supplementary Figure 1. Enforcing IRX1 on gastric cancer cells suppresses VM Plating 1.5 105 cells of SGC-7901, SGC-7901/vector and SCG-7901/IRX1 on the 3D gel, after 4-day culture without changing culture media, the cells were ﬁxed with 4% formaldehyde in phosphate-buffered saline (PBS) for 10 min and stained with PAS. The formation of VM was assessed as PAS-positive extracellular matrix (ECM) surrounded by cancer cells. SGC-7901/IRX1 cells revealed strong inhibiting effect on VM formation whatever in tubular length and tubular intersecting nods, compared with mock or controls (Figure 4). Enforcing IRX1 on gastric cancer cells suppresses angiogenesis in chick embryo A traditional chick embryo chorioallantoic membrane (CAM) assay was used for observing the effect of restoring IRX1 on angiogenesis. A 30-ml supernatant from SGC-7901, SGC-7901/vector and SCG-7901/IRX1 was dropped to the surface of the CAM daily, which covered a piece of sterilized ﬁlter paper disc. After 3-day treatment, the eggs were photographed, and the vessel numbers around the ﬁlter paper disc were assessed. The blood vessel numbers treated with SCG-7901/IRX1 supernatant were signiﬁcantly reduced, compared with mock or controls (6.41±2.59 vs 9.18±1.99 and 10.27±2.64, P ¼ 0.001; Figure 5). Enforcing IRX1 on gastric cancer cells suppresses cell-ECM adhesive ability A 150-ml cell suspension of SGC-7901 (1.0 106 /ml) was added to the wells coated with ﬁbronectin or collagen IV and incubated for 60 min. After cell staining and washing, we examined the attached cells in the micro- titer plate. The OD (optical density) value at 560 nm represents the cell-ECM adhesive ability. The OD 560 value of SGC-7901/IRX1 cells was signiﬁcant lower than that in mock or controls on type IV collagen assay (0.576±0.043 vs 0.653±0.059, 0.717±0.061, P ¼ 0.002). The OD 560 value of SGC-7901/IRX1 cells was signiﬁcant lower than that in mock or controls on ﬁbronectin assays too (0.682±0.040 vs 0.813±0.005, 0.870±0.040, Po0.001). IRX1 can downregulate BDKRB2 or its downstream effector PAK and inﬂuence multiple biological behaviors Our previous cDNA microarray and chromatin im- munoprecipitation implied that BDKRB2 functions as one of downstream targets of IRX1 transcription factor. We transfected IRX1 to SGC-7901 cancer cells and found that BDKRB2 expressing level was signiﬁcantly decreased by RT–PCR and western blot (Figure 6a). We knocked down BDKRB2 by gene-speciﬁc small Oncogene