A 96-well plate was precoated with an oligonucleotide containing the NF-κB p65 binding consensus site. The active form of the p65 subunit was detected using antibodies specific for an epitope that was accessible only when the appropriate subunit bound to its target DNA. An HRP-conjugated secondary antibody provided a colorimetric readout that was quantified by a spectrophotometer (450 nm). Statistical analysis Data were analyzed using SPSS software (version 16.0). Results were expressed as the mean ± SD. Statistical analysis was performed by one-way ANOVA and Student’s t-test. P < 0.05 was considered statistically significant. Results Effects of

HUVECs on the tumorigenicity of MHCC97H cells in vivo To assess the effects MI-503 concentration of HUVECs on the tumorigenicity of HCC cells, we injected subcutaneously MHCC97H cells into nude mice either alone or in combination with HUVECs. Subcutaneous tumors developed at the site of implantation in mice. The tumor size in mice implanted with a mixture of HUVECs and MHCC97H cells were much larger than that in mice implanted with MHCC97H cells alone (Figure 1A). In addition, the expression of HCC invasion/metastasis-associated genes (MMP2,

MMP9, OPN, and CD44) in the subcutaneous mixed tumor of MHCC97H cells and HUVECs were significantly higher than those formed by MHCC97H cells alone (*p < 0.05; Figure 1B). Figure 1 Subcutaneous tumorigenicity Microbiology inhibitor test of MHCC97H cells premixed with HUVECs and the expression of HCC invasion/metastasis-associated

genes. (A) MHCC97H cells as well as a mixture of MHCC97H cells and HUVECs were subcutaneously implanted Phosphoglycerate kinase into nude mice as described in the “Material and methods” section. Representative tumors resected from nude mice appeared 10 days after implantation. (B) The expression of MMP2, MMP9, OPN, and CD44 were detected by qRT-PCR in subcutaneous tumors (*P < 0.05, **P < 0.01, ***P < 0.001 vs. MHCC97H cells alone). Changes in the malignant properties of HCC cells under CM stimulation As shown in Figure 2A and B, the proliferation of HCC cells treated with CM derived from HUVECs significantly increased compared with that treated with EBM (*p < 0.05). The numbers of nuclear Ki67-positive cells in the MHCC97H cells treated with CM also increased. These results supported that some secreted factors derived from HUVECs may stimulate HCC cell proliferation in vitro. Figure 2 Changes in the malignant properties of HCC cells under CM stimulation. (A) CM significantly promoted HCC cell proliferation (**P < 0.01 vs. EBM at 48 h) was measured by CCK8. (B) The expression of Ki67 in the nucleus of HCC cells. (C) Wound healing assays were performed with MHCC97H cells incubated by CM or EBM. The amount of migrating cells at the wound front was much higher than that in the control (**P < 0.01).