VEGFA Upregulates FLJ10540 Figure 4. VEGF-A promotes FLJ10540 protein expression and enhances FLJ10540-induced migration and invasion in lung cancer cells. (A) The microarray expression patterns of VEGF-A and FLJ10540 in lung adenocarcinoma patients were shown. The results were normalized against the expression patterns of 56 chips (HG_U133A). N: adjacent non-tumor tissues; T: tumor tissues. (B, left panel) VEGF-A induced an increase in FLJ10540 protein levels in a dose-dependent manner. After treatment with various concentration of VEGF-A (left panel) or VEGF-B (right panel) for 10 min in CL1-0 cells, the total proteins were extracted from CL1-0 cells and probed with antibody against FLJ10540. (B, middle panel) Serum-starved CL1-0 cells were pre-treated with or without various concentrations of SU5416 for 2 hours; cells were then stimulated with 20 ng/ml VEGF-A for 10 min. b-actin was used as an internal loading control. (C) Serum-starved CL1-0 cells were pre-treated with or without SU5416 for 2 hours; cells were then stimulated with VEGF-A (at the concentration of 20 ng/ml) for 3 hours. The migration and invasion relative-folds were normalized against vehicle cells. (D, left, 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 and allowed to adhere for 12 hours, and were then incubated with or without VEGF-A (20 ng/ml) for 3 hours. At the end of the assay, the cells on the topside were scraped, and the cells that migrated to the bottom were fixed and stained with Giemsa. (D, left, bottom panel) For the invasion assay, 16104 cells were seeded after Matrigel was added, and were then incubated with or without VEGF-A (20 ng/ml) for 3 hours. All of the data represent the mean6s.d. of three independent experiments. (E) Indirect immunofluorescence analysis of FLJ10540 in VEGF-A-treated cells. The protein expression and the subcellular localization of FLJ10540 were analyzed in the presence or absence of VEGF-A (20 ng/ml) in CL1-0 cells using immunofluorescence microscopy. After being incubated with or without VEGF-A for 30 min or 180 min, the cells were fixed with 3.7% formaldehyde and processed for indirect immunofluorescence microscopy. FLJ10540 translocated to the cell membrane upon VEGF-A treatment (Arrowhead). Bar: 10 mm. doi:10.1371/journal.pone.0005052.g004 PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5052