higher in the highly invasive CL1-5 cells than in the low invasive CL1- 0 cells, as shown by western blot analysis (Figure 1C, right panel). The results indicated that the upregulation of FLJ10540 mRNA and protein expression was positively correlated with the invasive capability of the lung cancer cells, raising the possibility that upregulation of FLJ10540 might lead to some of the abnormalities found in human lung adenocarcinoma. Overexpression of FLJ10540 promotes cell migration and invasion Since FLJ10540 is overexpressed in lung adenocarcinoma and a highly invasive lung cancer cell line, it seemed possible that its expression might affect cell motility. To clarify role of FLJ10540 in motility in the mammalian cells, we examined whether overex- pression of FLJ10540 was able to affect cell migration and invasion. Two different cell types were employed to evaluate this possibility. First, CL1-0 cells stably expressing hemagglutinin (HA)- tagged FLJ10540 were established. Several stable transfectants with increasing levels of the FLJ10540 protein were selected (Figure 2A, left panel) for further studies. Interestingly, FLJ10540 overexpression in CL1-0 cells was associated with strikingly altered cell morphology. FLJ10540 overexpressed clones were decreased in cell size and appeared to be more spindle-shaped and had increased intercellular separation compared to vehicle control clones, which were round-shaped (Figure 2A, middle panel). VEGFA Upregulates FLJ10540 However, the proliferation rate, as determined by the MTT assay, was not significantly different from that of the vehicle control or cells expressing FLJ10540 during 24 hours (Figure 2A, right panel). To examine the effect of FLJ10540 on human lung cancer cell migration, stable cell lines expressing HA-FLJ10540 or vehicle control were seeded on Transwell chambers. The results showed that FLJ10540 CL1-0-stable transfectants could increase their migration, as measured by the Transwell migration assay; this was compared to the vehicle control, where very few cells migrated (Figure 2B, left upper panel). Quantitatively speaking, the migration ability of FLJ10540 CL1-0-stable transfectants was approximately 6–7 fold higher than that of the vehicle control (Figure 2B, right upper panel). We next carried out an in vitro invasion assay using a Matrigel coated barrier filter. After a 24 hour incubation in a Matrigel-coated Transwell chamber, FLJ10540 CL1-0-stable transfectants had invaded the basement membrane material and passed through the pores to reach the underside of the filter (Figure 2B, left bottom panel). Similarly, FLJ10540 CL1-0 expressing cells showed a 10–12 fold higher invasion ability than the vehicle control cells (Figure 2B, right bottom panel). Second, to avoid clonal effects, a retroviral-based Flag-tagged FLJ10540 was infected into an RK3E cell line (Figure S2A) that had previously been used successfully to evaluate migration or invasion induced by different genes and signaling pathways, such as fibroblast growth factor 9 (FGF9) [24], k-ras [25], and Snail Figure 2. FLJ10540 overexpression promotes lung cancer cell migration and invasion. (A, left panel) HA-tagged-FLJ10540 stable clones of CL1-0 cells were established. The cell lysates were subjected to immunoblot analysis with anti-HA antibody. (A, middle panel) Phase-contrast images of monolayer cultures of CL1-0 cells expressing FLJ10540 and vehicle control were shown. (A, right panel) To measure cell growth rates, 56103 cells of vehicle-CL1-0 and CL1-0-FLJ10540 stable clones were plated at day 0 in 96-well plates with 10% FBS. The cells were counted at indicated time points by the MTT assay (OD570) to quantify the cell growth. Data were normalized against the OD570 value on day 0. The growth rate of CL1-0 cells is shown as the mean6s.d. of three independent experiments. (B, upper panel) For the migration assay, 56103 cells of vehicle-CL1-0 and CL1-0-FLJ10540 stable clones were seeded into the top of a Transwell insert. After 24 hours, the cells on the topside were scraped, and the cells that migrated to the bottom were fixed and stained with Giemsa. The migration relative-fold of vehicle-CL1-0 and CL1-0-FLJ10540 stable clones was normalized with vehicle control and presented diagrammatically. (B, bottom panel) For the invasion assay, 16104 cells were seeded after Matrigel was added. The invasion relative- fold of stable clone was normalized against vehicle cells and represented diagrammatically. All of the data represent the mean6s.d. of three independent experiments. doi:10.1371/journal.pone.0005052.g002 PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5052