MTT assay for cell growth Vehicle-CL1-0, FLJ10540-CL1-0 stable clones, vehicle-negative control and siRNAs FLJ10540 transfected cells of CL1-5 or H1299 (56103 ) were seeded in 96-well plates in RPMI containing 10% FBS for 24 h followed by MTT assay to quantify the cell growth. Data were normalized against OD570 value on day 0 of each vehicle control. Migration and Invasion Assay The migration and invasion assays of the CL1-0 stable clones or the RK3E infected cells were evaluated using a 24-well Transwell (8-mm pore size polycarbonate membrane, Costar) chambers. For the migration assay, 56103 CL1-0 stable clones were suspended in 400 ml of DMEM or RPMI containing 10% FBS and were seeded into the upper chamber, while 600 ml of DMEM or RPMI containing 10% FBS was added to the outer side of the chamber. After being cultured in a 37uC, 5% CO2/95% air environment and allowed to adhere for 12 hours and then incubated with or without VEGF-A (20 ng/ml) for 10 min, cells on the upper surface of the membrane were removed by a cotton tip applicator and migratory cells on the lower membrane surface were fixed by methanol and stained with Giemsa (Sigma). Cell migration values were determined by counting from three independent membranes and then normalized using vehicle cells to give a relative ratio. For invasion assay, 80 mg/ml of Matrigel (BD Bioscience) was added to the upper surface of the membrane and allowed for gelling at 37uC for overnight. Cells (16104) in 400 ml of DMEM containing 10% FBS were seeded to the upper chamber, and 600 ml of DMEM containing 10% FBS was added to the outer side of the chamber. The rest of procedures for invasion assay were the same as in the migration assay. Tissue Microarray Analysis Tissue cores manually removed from 273 archived lung adenocarcinomas were inserted into paraffin blocks to generate tissue arrays. The population includes 153 male and 120 female patients, with variable pathological stages (stage IA, 45 patients; IB, 105; IIA, 5; IIB, 18; IIIA, 49; IIIB, 21; IV, 12). Sections 5 mm in thickness were obtained and mounted on positively charged slides for immunohistochemical studies. Immunohistochemistry Immunohistochemical detection of protein expression is per- formed on formalin-fixed, paraffin-embedded tissue sections of lung adenocarcinoma tissue arrays with standard procedures. The tissue sections were incubated at 4uC overnight with primary antibodies for VEGF-A (1:800 dilution, BioGenex, sc-152), FLJ10540 (1:800 dilution) and AKT473-p (1:100 dilution). The intensities of the immunohistochemical staining were scored based on the following criteria: no or minimal staining in ,10% of the tumor cells, score 0; faint, barely visible staining in .10% of the tumor cells, score 1; weak to moderate staining in .10% of the tumor cells, score 2; strong staining in .10% of the tumor cells, score 3. Statistical analyses The correlation between each paired IHC scores of AKT473-p, VEGF and FLJ10540 were analyzed by Spearman’s rank tests. A p value ,0.05 was considered statistically significant. All analyses were carried out using statistics software SPSS 12.0 edition. Immunofluorescence microscopy CL1-0 cells were incubated with VEGF (20 ng/ml) for 30 min or 180 min, the cells were washed with PBS and fixed with 3.7% VEGFA Upregulates FLJ10540 formaldehyde in PBS for 15 min at 25uC. Cells on the coverslips were blocked with 5% BSA (Sigma) in PBS. The fixed cells were probed with anti-FLJ10540 antibody (1:400) for 12 hrs at 4uC and then washed three times with 0.1% Triton X-100 in PBS. Immunofluorescent cell images were acquired using an Olympus Fluoview FV1000 Confocal laser scanning microscope (Olympus). Images were processed with Adobe Photoshop software (Adobe Systems). All data were collected during the same session to avoid potential differences due to fading. Supporting Information Figure S1 Oncology. Molecular portraits of lung adenocarcino- ma. (A) Supervised hierarchical clustering showed the 826 (634 down- and 192 up-regulated) transcript expression patterns of 26- pairwise lung adenocarcinoma samples. The results were shown in a dendrogram format, in which rows represent individual transcripts and columns represent tissue samples. Especially, FLJ10540 was shown in one of 192 up-regulated transcripts. The color in each cell reflected the expression level of the corresponding tissue, relative to its median expression level. The scale extends from fluorescence ratios of 0.25 to 4 relative to the median level for all samples. (B) The mRNA expression level of FLJ10540 was determined by Q-RT-PCR in 16 lung cancer patients. Overexpression of FLJ10540 was observed in 15 out of 16 lung cancer patient samples. The results were normalized against the expression level of GAPDH mRNA in each sample. Found at: doi:10.1371/journal.pone.0005052.s001 (8.38 MB TIF) Figure S2 Oncology. FLJ10540 overexpression enhances motil- ity of RK3E cells. (A) FLJ10540 stable clones of retrovirus infected Flag-tagged FLJ10540 in RK3E cells was established. The cell lysates were subjected to immunoblot analysis with anti-Flag antibody. (B) For the migration assay, 56103 cells of vehicle- RK3E and RK3E-FLJ10540 infected cells 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 photography results of vehicle-RK3E and RK3E-FLJ10540 infected cells are shown (2006). The migration relative-folds of vehicle-RK3E and RK3E- FLJ10540 infected cells were normalized with vehicle control and presented diagrammatically. (C) For the invasion assay, 16104 cells were seeded after Matrigel was added. The invasion photography results of vehicle-RK3E and RK3E-FLJ10540 infected cells are shown (2006). The invasion relative-folds of stable clone and infected cells were normalized against vehicle cells and represented diagrammatically. All of the data represent the mean6s.d. of three independent experiments. Found at: doi:10.1371/journal.pone.0005052.s002 (9.12 MB TIF) Figure S3 Oncology. FLJ10540 mediates cell migration and invasion through the PI3K/AKT signaling pathway. (A). The migration and invasion photography results of vehicle-CL1-0, CL1-0-FLJ10540 stable clones, vehicle-RK3E and RK3E- FLJ10540 infected cells were treated with or without LY294002 at the final concentration of 10 mM are shown (2006). The data represent the mean6s.d. of three independent experiments. (B) Vehicle-CL1-0 and CL1-0-FLJ10540 stable clones on the Transwell insert were serum-starved for 24 hours and treated with or without the indicated inhibitors for 2 hours. Cells were then stimulated with or without VEGF-A at the final concentra- tion at 20 ng/ml for 10 min. The migration and invasion ratios of vehicle-CL1-0 and CL1-0-FLJ10540 stable clones were deter- mined. The data represent the mean6s.d. of three independent experiments. PLoS ONE | www.plosone.org 11 April 2009 | Volume 4 | Issue 4 | e5052