[26]. RK3E cells exhibit multiple features of epithelia, such as a near-normal karyotype and a low background of cell migration and invasion. Mixed pools of infected RK3E cells were used for the migration and invasion assays. Again, FLJ10540 expressing cells showed a 4–5 fold higher migration ability than the vehicle control cells (Figure S2B) and a 7 fold higher invasion ability than the vehicle control cells (Figure S2C). Taken together, these findings support the hypothesis that FLJ10540 can promote cell migration and invasion in mammalian cells. Knock-down of endogenous FLJ10540 by siRNA suppresses lung cancer cell migration and invasion To confirm that FLJ10540 affects cell migration and invasion in human lung cancer cells, we used a siRNA approach to inhibit endogenous expression of FLJ10540 and then assayed the migration and invasion abilities of CL1-5 and H1299 cells. First, to examine the specificity of siRNAs, an HA-tagged FLJ10540 expression construct was co-transfected with three different siRNAs; these consisted of two chemically synthesized siRNAs specific for FLJ10540 (siFLJ10540-1 and siFLJ10540-2) and a negative control siRNA, which were each transfected into HEK293T cells. Both FLJ10540 siRNAs showed almost complete inhibition of HA-tagged FLJ10540 expression, as shown by western blot analysis using an anti-HA antibody, while the level of HA-tagged FLJ10540 remained high with the negative control siRNA (data not shown). Second, to examine if the specific FLJ10540 siRNAs could inhibit endogenous FLJ10540 expression in CL1-5 and H1299 cells, we transfected siRNAs into CL1-5 and H1299 cells for 24 hours, followed by western blot analysis using an antibody against FLJ10540. The data showed a dramatic reduction in the FLJ10540 protein levels with both FLJ10540 siRNAs, and there was no significant reduction of FLJ10540 when the negative siRNA was used (Figure 3A and B, left panel). However, the proliferation rate, as determined by the MTT assay, was not significantly different between the negative control cells and cells expressing FLJ10540 siRNAs during 24 hours (Figure 3A and B, middle panel). Third, to study the effect of siRNA FLJ10540 transfection on migration, negative control and siRNA FLJ10540-transfected CL1-5 or H1299 cells were separately seeded on Transwell chambers with uncoated filters. After 24 hour incubation, the motility potential of siRNA FLJ10540 cells was found to have been strongly inhibited by the siRNAs (Figure 3A and B, right panel). Finally, the same panels of negative or FLJ10540 siRNA transfected cells were seeded in a Matrigel-coated Transwell chamber. After 24 hours of incubation, the invasive potential of the siRNA FLJ10540 cells appeared to be significantly reduced (Figure 3A and B, right panel). These results strongly support the idea that FLJ10540 plays a role in cell migration and invasion in human lung cancer cells. Using the concept of syn-expression to uncover VEGF-A, but not VEGF-B, as the upstream regulator of FLJ10540 Genes belonging to the same pathway often show the same expression profiles, which is referred to as syn-expression [27]. Hence, to map the potential signaling pathway or upstream regulator(s) participating in FLJ10540-elicited migration and invasion characteristics, we employed our lung cancer microarray dataset, as described earlier, to gain insight into the functional concordance of co-expressed genes. To test this hypothesis, we first examined whether the mRNA expression profile of FLJ10540 correlates with any ligand or receptor in our lung cancer microarray database (Figure 1). Of these co-expressed genes, the VEGFA Upregulates FLJ10540 top candidate is VEGF-A, which was highly positively correlated with the mRNA expression level of FLJ10540 in paired lung cancer patients (r = 0.7 p,0.001) (Figure 4A). VEGF-A is known to be a critical pro-angiogenic factor, with abilities to regulate many steps in the angiogenic process, including proliferation, migration, invasion, and survival [28,29]. In fact, VEGF-A is highly expressed in many malignant tumors, including lung cancer [30,31,32], and the expression of VEGF-A is associated with poor prognosis [33,34] and the presence of lymph node metastasis [35], raising the possibility that the biological roles of FLJ10540 and VEGF-A might be functionally linked in lung adenocarcinoma. Given the major role of VEGF-A in regulating migration and invasiveness, we were first interested in whether VEGF-A could modulate FLJ10540 protein expression. The results showed that the protein expression level of FLJ10540 was upregulated in a VEGF-A dose-dependent manner in CL1-0 lung cancer cells (Figure 4B, left panel). We next asked whether the antagonistic effect of SU5416, a VEGFR-2 tyrosine kinase inhibitor, on biological activities of VEGF-A could be attributed to the inhibition of FLJ10540 protein overexpression induced by VEGF-A in lung cancer cells. The results demonstrated that upregulation of FLJ10540 in the presence of VEGF-A was inhibited by simultaneous addition of SU5416 in a dose-dependent manner (Figure 4B, middle panel). On the contrary, the protein level of FLJ10540 was not affected by treatment with VEGF-B (Figure 4B, right panel). In fact, FLJ10540 does not share similar expression patterns with VEGF-B, at least not in our lung cancer microarray, supporting the idea that synexpression patterns could be used to infer the function of unknown genes [27]. VEGF-A-induced FLJ10540 overexpression and translocation enhances lung cancer cell migration and invasion through an EMT-independent pathway To test the biological significance of VEGF-A-induced upregu- lation of FLJ10540, we examined the influence of FLJ10540 on migration and invasion in the presence or absence of VEGF-A. First, we examined whether parental CL1-0 cells could display cell motility after stimulation by VEGF-A. In figure 4C, CL1-0 cells exhibited more migration and invasive abilities in the presence of VEGF-A alone than VEGF-A combined with SU5416 or vehicle alone, suggesting that the addition of VEGF-A significantly increased the migration and invasion of CL1-0 cells, which was paralleled by an increase in FLJ10540 levels. Similar results were found in FLJ10540 CL1-0-stable transfectants, with or without VEGF-A stimulation (Figure 4D). To test whether the subcellular localization of FLJ10540 could be altered in lung cancer cells upon VEGF-A stimulation, we adopted the indirect immunofluorescence approach to observe FLJ10540 localization in CL1-0 lung cancer cells either with or without VEGF-A stimulation. As shown in figure 4E, VEGF-A treatment not only resulted in an increase in FLJ10540 protein expression, but it also promoted the translocation of a fraction of FLJ10540 to the cell membrane. In addition, the differences in cell morphology were more apparent, highlighting the extension of VEGF-A treated cells (Figure 4E, 30 min and 180 min) as compared with the rounded CL1-0 cells (Figure 4E, 0 min). These morphological changes prompted us to examine the phenomenon of epithelial-mesenchymal transition (EMT). During tumorigene- sis, EMT may increase the motility and invasiveness of cancer cells, and malignant transformation may be associated with signaling pathways promoting EMT [36]. A previous study also indicated that extracellular signals induced by VEGF-A are strongly implicated in EMT in human pancreatic carcinoma cells [37]. To examine whether FLJ10540 might mediate VEGF-A- PLoS ONE | www.plosone.org 5 April 2009 | Volume 4 | Issue 4 | e5052