BMC Cancer 2007, 7:95 http://www.biomedcentral.com/1471-2407/7/95 plasm but only some of the nucleus in ACCs cells. The mean rate of NF-κB p65 nuclear staining detected in ACC- M and ACC-2 was 30.35 ± 2.52% and 17.97 ± 1.50%, respectively. The rate of NF-κB p65 nuclear localization in ACC-M was significantly higher than that in ACC-2 (P < 0.01). The red fluorescence detected in the cytoplasms of ACCs cells is the VEGF staining. The mean immunofluorescence intensities of VEGF in ACC-M and ACC-2 cell lines were 38.98 ± 4.98 and 24.10 ± 1.57, respectively (Figure 3). The expression level of VEGF in ACC-M was significantly higher than that in ACC2 (P < 0.01, Figure 3B). The Spearman Correlation analysis was performed to quantitate the association between two variables. The expression level of VEGF was significantly correlated to NF-kB p65 nuclear localization in both of the ACC-M and ACC-2 cell lines (P < 0.01). Down regulation of VEGF mRNA expression by inhibition of NF-κBp65 activation ACC-M and ACC-2 cells were transfected with the mutant IκBα expression vector. The constitutive NF-κBp65 activ- ity found in ACC-M and ACC-2 cell lines was completely abolished in ACC-M/IκBαM and ACC-2/IκBαM cell lines (figure 1, lane 3–4). Furthermore, the constitutive mRNA expression level of VEGF was effectively inhibited by over- expression of IκBαM in ACC-M/IκBαM and ACC-2/ IκBαM cells (figure 2, lane 3–4). ACCs cell lines enhanced the migration of endothelial cells After 24 hours of culture, the scrape-wounded UVECs monolayers incubated with medium from ACC-M showed that cells migrated into the denuded area more densely than the ones incubated with medium from ACC- 2 (Figure 4b, c). On the other hand, in the medium from control cultures, the cells sparsely migrated into the denuded area and the wound remained open (Figure 4a). ACCs cell lines stimulated the tube formation of endothelial cells In the presence of medium from ACC-M, UVECs formed organized elongated tube-like structures resembling capil- laries with an extensive network (Figure 4f). While in the presence of medium from ACC-2, the formation of the tubular-like structures and networks by UVECs were decreased (Figure 4e). However, cultured with medium from the control, tube formation was diminished and no such well-organized structures were observed (Figure 4d). The results indicated that ACCs cells with high metastasis ability could stimulate the tube formation of angiogen- esis. ACCs cell lines stimulated the UVEC sprouting into the gels matrix After a week of culture with medium from ACC-M in the outer well, the tubules structure grew further thickening FMiiggurarteio4n and tube formation of endothelial cells Migration and tube formation of endothelial cells. UVECs were cultured with the conditioned medium from control (a), ACC-2(b) and ACC-M (c) for migration assay. Edges of the denuded areas are marked by dashed lines. UVECs were cultured with the conditioned medium from control (d), ACC-2(e) and ACC-M (f) for tube formation assay. One of tube-like structure was marked by asterisk (*) in figure b and c. into the gel and the monolayer on surface of the gel grad- ually reach sub-confluence. At the thickened branch points of the tubules structure, the UVECs sprouting into the gel matrix could be observed by the confocal laser scanning microscope (Figure 5c). The mean thickness of sprouting was 53.96 um (Figure 5f). While cultured with the medium from ACC-2, there were less UVECs sprout- ing into the gel matrix (Figure 5b) and the mean thickness of sprouting was 21.76 um (Figure 5e). It showed that UVECs cultured with ACC-M (figure 5f) presented more cell sprouting points and thicker sprouting layers than cul- tured with ACC-2 (figure 5e). On the other hand, UVECs cultured with medium from the control only formed IcFneiglvlsuitroe t5hree-dimensional angiogenic assay of endothelial In vitro three-dimensional angiogenic assay of endothelial cells. Control (figure 5a), ACC-2 (figure 5b) and ACC-M (fig- ure 5c) induce UVECs sprouting into the gel matrix (white arrow) and the mean thickness of sprouting was showed by figure 5d, figure 5e and figure 5f. UVECs cultured with ACC- M (figure 5f) shows more cell sprouting points and thicker sprouting layers than cultured with ACC-2 (figure 5e). Page 5 of 7 (page number not for citation purposes)