Subcutaneous injection of tumor cells. (a) HepG2-VASH2, HepG2-wt or HepG2-shVASH2 cells were suspended at a density of 107 cells/ml, and 100 μl was bilaterally injected into the flank of nude mice (n=5). After 20 days the tumors were removed. The tumorigenesis rate was 100% for each group. The HepG2-shVASH2 tumors were smaller than those of the other groups, whereas the size of the HepG2-VASH2 tumors was not significantly different than that of the HepG2-WT tumors. (b) Tumor growth curves. After injection with tumor cells, the length and width of tumors were measured using vernier calipers every 4 days. The volume of the tumors was calculated using the equation volume=1/2 × length × width2. The data are presented as the average±s.d. of 10 tumors for each group. *A significant difference between the groups when compared with the HepG2-shVASH2 group (P<0.05). (c) Three tumors from each group were resected, mRNA was extracted and VASH2 expression was measured using qRT–PCR. (d) CD31 immunohistochemistry was performed on the tumor tissue from the HepG2-VASH2, HepG2-WT and HepG2-shVASH2 groups. All scale bars represent 200 μm; magnification, × 20. (e) Microvessel density of CD31 immunohistochemistry. Each slide was evaluated with three fields, and data were analyzed as mean vessel number of these three fields.