BMC Cancer 2007, 7:95 http://www.biomedcentral.com/1471-2407/7/95 Statistical evaluation The experiments were repeated thrice and all data are pre- sented as mean ± S.D. Statistical analysis was performed by ANOVA test. The Spearman rank correlation coefficient test was applied for the correlation among the expression of NF-κB and VEGF. P-values less than 0.05 were consid- ered to be significant. Results NF-κBp65 activity and mRNA expression level of VEGF in ACCs cell lines NF-κBp65 DNA binding activity in the nuclear extracts from ACC-M and ACC-2 cell lines was showed in Figure 1, lane 1–2. There was a constitutive NF-κBp65 activity in ACC-M and ACC-2 cell lines. In the ACC-M, the intensity of the shift band of p65/P50 was much stronger than that in ACC-2. Figure 2 showed the mRNA expression band of VEGF in ACC-2 and ACC-M. The mean level of VEGF mRNA in ACC-M (0.575 ± 0.10) was about 2-fold than that in ACC- 2 (0.309 ± 0.11), and the statistical difference was deemed significant (P < 0.01) [see additional file 1]. Nuclear localization of NF-κB and semi-quantity immunofluorescence of VEGF in ACCs cell lines As Figure 3A showed, the green fluorescence is the NF-κB p65 staining, which was detected in almost all of the cyto- FIEκMiBgSαuAMres,hA1oCwCed-2thanedacAtCiviCty-2o/fIκNBFα-MκBcpel6l5li ninesACC-M, ACC-M/ EMSA showed the activity of NF-κB p65 in ACC-M, ACC-M/ IκBαM, ACC-2 and ACC-2/IκBαM cell lines. Arrows indicate the migration of the induced NF-κB DNA-binding com- plexes. Migration of the free probe is not shown. The Oct-1 motif was used as a control for quality and quantity of cell extract. FRAiTCg-CuPrC-2eR,2AshCoCw-eMd/ItκhBeαmMRaNnAd AexCpCre-2ss/IiκoBnαoMfVcEeGll Fli ninesACC-M, RT-PCR showed the mRNA expression of VEGF in ACC-M, ACC-2, ACC-M/IκBαM and ACC-2/IκBαM cell lines. IcVFmoEignmGufouFrcoeeafxl3uploarsreesrscesedcnaicnnenAidnCoguCabn-lMaelysastniasdinsiAhnoCgwCaen-d2 NsceeFml-l κil-iBnqeupsa6n5titaantdive Immuofluorescence double staining and semi-quantitative confocal laser scanning analysis showed NF-κB p65 and VEGF expressed in ACC-M and ACC-2 cell lines. As figure 3A showed, the rate of NF-κB p65 nuclear localization (a) (white arrow) and VEGF staining intensity (b) in ACC-M was higher than that in ACC-2 (e) and (f). As figure 3B shows, bars represent the mean value of immunofluorescence inten- sity of VEGF and nuclear staining rate of NF-κB p65 in two cell lines, P < 0.01(*). Page 4 of 7 (page number not for citation purposes)