Wang et al. BMC Cancer 2010, 10:415 http://www.biomedcentral.com/1471-2407/10/415 Page 5 of 11 Figure 1 Expression of TLR9 in glioma cell lines and glioma samples. A. mRNA expression of TLR9 (a) Expression of TLR9 in human U87 glioma cell line(lane1) and human U251 glioma cell line(lane2); (b) Expression of TLR9 in rat glioma cell line C6. (c) Expression of TLR9 in 34 clinical glioma samples (lane1-15, Grade IV gliomas; lane16-28, grade III gliomas;lane 29-34, grade II gliomas). GAPDH was used as positive control. B. Protein expression of TLR9 in human glioma cell lines U87 and U251 with immunofluorescence staining. cytoplasmic localization of TLR9(red fluorescence, Cy3) in U87 and U251 was confirmed by confocal microscopy, nucleus was stained with Hoechst(Blue fluorescence). (a) TLR9 staining in U87 cells. (b) Hoechst staining of nucleus in U87 cells. (c) The combined figure of a and b. (d) negative control, staining of U87 cells with secondary antibody and Hoechst, but without primary anti-TLR9 antibody. (e) TLR9 staining in U251 cells. (f) Hoechst staining of nucleus in U251 cells. (g) The combined figure of e and f. (h) Negative control, staining of U251 cells with secondary antibody and Hoechst, but without primary anti-TLR9 antibody. dead before July 2009) using univariate Cox regression analysis(Table 3), we found that the expression levels of TLR9 protein were correlated with PFS of GBM patients (p = 0.013). As shown in Table 3, the multivariate analy- sis revealed that the expression levels of TLR9 protein level was a significant prognostic factor in GBM patients, independent of the conventional clinical vari- ables (hazard ratio [HR], 3.205; 95% confidence interval [CI], 1.152-8.920; p = 0.026). In this study, age, gender, preoperative KPS, and extent of resection were not asso- ciated with PFS in GBM. We assessed the discriminative value of expression levels of TLR9 (low expression and high expression) for survival of patients with GBM using Kaplan-Meier analysis. We found that the expres- sion levels of TLR9 could significantly discriminate the survival of the two GBM subgroups, with a mean PFS of 9 months (median PFS, 11.108 months; 95% CI, 8.843-13.372) for low TLR9 expression group versus mean 6 months (median PFS, 6.958 months; 95% CI, 5.634-8.283) for high TLR9 expression group (p = 0.001) (Fig. 2B and Table 3). CpG-ODN has no effect on glioma proliferation in vitro MTT assay were performed to investigate the effect of CpG ODN on the proliferation of glioma cells, glioma cell lines U87, U251 and C6 were used in this analysis, our results showed that CpG-ODN (ODN 2006) did not influence the proliferation rate of the three glioma cell line tested within 72 hours (Fig. 3), when compared with non CpG ODN (ODN2006 control) or PBS control. The ODN 2006 used in this research has been reported to be efficient activators of cells displaying mRNA expression of TLR9 [20], and was widely used as a adju- vant for immunotherapy.