VEGFA Upregulates FLJ10540 1 and siFLJ10540-2) were transfected into CL1-0 cells for 24 hours. After transfection, cells were treated with or without VEGF-A (20 ng/ml) for 10 min. Western blotting was performed as in (C). (E upper panel) Serum-starved CL1-0 cells were pre-treated with or without SU5416 for 2 hours; cells were then stimulated with or without 20 ng/ml VEGF-A for 10 min. Cell lysates were immunoprecipitated with polyclonal antibodies against p110-a or protein IgG (as a control), which was followed by immunoblotting with p110-a, p85-a, and FLJ10540. (E lower panel) A negative control siRNA and two different FLJ10540 siRNAs (siFLJ10540-1 and siFLJ10540-2) were transfected into CL1-0 cells for 24 hours. After transfection, cell lysates were immunoprecipitated with polyclonal antibodies against p110-a, p85-a, or protein IgG (as a control), followed by immunoblotting with p110-a, p85-a, and FLJ10540. doi:10.1371/journal.pone.0005052.g006 several lung cancers and in pre-invasive bronchial lesions, implicating the PI3K/AKT pathway in lung cancer development [44]. Several groups have reported that amplification, mutation, and high levels of phosphorylated AKT in tumor tissues from lung cancer patients correlated with poor prognosis in lung cancer patients in stage I of the disease or with primary tumors [42,44,45,46,47]. Moreover, in a panel of lung cancer cell lines, PI3K-dependent AKT phosphorylation and activation of PI3K also correlated with resistance to chemotherapy and radiotherapy [48,49]. In short, inhibition of PI3K/AKT activation and disruption of the associated proteins that enhance PI3K complex formation have been widely proposed to represent promising therapeutic approaches for treatment of lung cancer. In summary, the explosion in microarray studies has promised to shed light on potential disease markers and drug targets. This study illustrates that FLJ10540 is highly expressed in human lung adenocarcinomas, as well as in invasive lung cancer cell lines. This abnormal expression of FLJ10540 in mammalian cells is able to enhance cell migration and invasion. Moreover, the quantity of FLJ10540 in cells could serve as a critical mediator of VEGF-A- dependent biological events through activation of the PI3K/AKT signaling pathway, resulting in the progression of lung adenocar- cinoma. In short, FLJ10540 could represent a potential target for the development of novel therapies for this widespread type of cancer. Materials and Methods Patients and tumor samples A total of 56 samples from our previous study [50] were used for microarray analysis. These include pairwise samples from 26 patients, who underwent surgery for lung cancer at the Taipei Veterans General Hospital, and 4 lung cancer cell lines CL1-0, CL1-1, CL1-5 [51] and CL1-5-F4 [52]. The data have been deposited in NCBIs Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE7670. No patients had previously received any neoadjuvant treatment, e.g. chemotherapy, before the surgery. The study protocol had the approval of the ethics committee at Taipei Veterans General Hospital. All patients gave informed consents and signed the consent form individually. Study samples, including tumor and adjacent normal tissues, were obtained during diagnostic biopsy, and adjacent normal tissues were derived from neighboring site outside of the tumor. Both tumor and adjacent non-tumor tissues for subsequent Q-RT-PCR studies were confirmed by patholo- gists. Quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) To confirm the microarray data for FLJ10540, 16-pairs tumor and adjacent non-tumor tissues of lung patients’ samples were used to validate the mRNA expression level of FLJ10540 by using Q- RT-PCR, which was performed using a Taq-Man probe (ABI). Data were represented as mean6s.d. To analyze the distribution of tumor and adjacent non-tumor parts we performed the Wilcoxon signed rank test between two groups for statistical analysis. A P value of less than 0.05 was significant. GAPDH (ABI) was used as an internal control for comparison and normalization the data. Assays were performed in triplicate using Applied Biosystems Model 7700 instruments. Preparation of retroviral stock and infection Retroviral vector-derived plasmid (1 mg) was introduced into 106 DF-1 cells by FuGENE 6 as described previously [53]. Viral supernatant produced from these cells were harvested. For viral infection, actively dividing RK3E/tv-a-expressing cells or control cells were seeded. The next day, cells grown in fresh growth medium were mixed with viral supernatant to a total volume of 2.5 ml for 60 mm-plate and incubated at 37uC in 5% CO2 incubator for one hour. Afterwards, fresh growth medium was then added to the cell/virus mixtures. Cell culture, transient transfection and establishment of stable clones All cell culture related reagents were purchased from Gibco-BRL. MDCK (clone 3B5) and RK3E cells were grown in DMEM medium containing 10% heat-inactivated FBS, 100 unit/ml penicillin and streptomycin (Gibco-BRL). CL1-0, CL1-5 and H1299 cells were grown in RPMI medium containing 10% heat-inactivated FBS, 100 unit/ml penicillin and streptomycin (Gibco-BRL). Transient transfection of HA-tagged FLJ10540 to CL1-0 was performed with LipofectamineTM (Gibco-BRL) according to the manufacturer’s instructions, whereas RK3E cells were infected with adenoviral Flag- tagged FLJ10540. CL1-0 cells stably expressing constructs were selected with 800 mg/ml G418 (Calbiochem). Individual clone was picked up and analyzed for exogenous FLJ10540 expression by western blotting. Each selected stable clones was lysed with extraction buffer as described below. Two double-stranded synthetic RNA oligomers (Ambion) (59-GGGAGAAATTGCACACTTA-39 and 59-GGACTTTTAGCAAAGATCT-39), deduced from human FLJ10540, and one Silencer negative control #4611G siRNA (Ambion) were used for siRNA experiments. Preparation of cell extracts, immunoprecipitation and western blot analysis Cell-free lysates were prepared as described earlier [54]. Briefly, cells were harvested, washed with PBS, and lysed in extraction buffer (20 mM PIPES, pH 7.2, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 1 mM dithiothreitol, 1 mM phenylmethyl- sulfonyl fluorid (PMSF) and 1 mM Na3VO4,). For immunoprecip- itation, 1 mg of total cell lysate was incubated with antibodies against target proteins and Protein A/G-agarose beads (Oncogene Research Product) to immunoprecipitate the target protein. Antibody against FLJ10540 (Abnova), AKT, AKT473-p (Cell Signaling), Flag (Sigma), HA (3F10, Roche), p110-a (Santa Cruz), p85-a (Santa Cruz) and b- actin (Sigma) were incubated with the membrane at room temperature for 1 h. The resulting IgGs were detected by incubation with secondary antibodies conjugated to HRP, and developed using the Western Lighting reagent. PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5052