[35,36]. The possibility of a BMP-independent proliferative mechanism for Gremlin is given credence by recent in vitro work by Mitola et al and Stabile et al [37,38]. Mitola et al demonstrate that Gremlin can bind the VEGF receptor-2 (VEGFR2) in a BMP- independent manner. This binding activates VEGFR2 in endo- thelial cells, culminating in a VEGFR2-dependent angiogenic response in vitro and in vivo. In addition, Costello et al [39] identified Gremlin as a highly significant member of a 90 gene cohort selectively upregulated in hypoxic primary human pulmo- nary microvascular cells. We tested whether increased normal lung cell growth induced by GREM1 transfection was BMP2-de- pendent. MRC-5 fibroblast cells with GREM1 transfection were incubated with BMP2. Addition of high-concentration BMP-2 had no effect on the growth of GREM1-transfected cells (Fig. S2). This suggests that GREM1 increases cell growth through a BMP- independent pathway and further study will be required to better elucidate the mechanism for the tissue-specific function of Gremlin. In summary, we present evidence that Gremlin is significantly overexpressed in human adenocarcinoma of the lung. Over- expression of Gremlin increases proliferation of both lung epithelial and fibroblast cell lines. Taken together, these studies suggest an oncogenic role for Gremlin in lung adenocarcinoma. Materials and Methods Ethics Statement The committee on Human Research (CHR) of UCSF has reviewed and approved the application for the collection of blood, sputum, and tissue samples from patients with suspected or biopsy- proven thoracic malignancies (approval number:10-03352). All samples have been collected under the IRB approval granted by the Committee on Human Research and written informed consents were obtained from all patients in this study. Patient Samples Fresh tumor tissue and adjacent normal tissue samples were obtained at the time of surgical resection of NSCLC patient tumors at the University of California, San Francisco (UCSF) between 1997 and 2007 and deposited in the Thoracic Oncology Tissue Bank. Patients were eligible for the study if they underwent surgical resection of NSCLC at UCSF with curative intent after January 1997. Samples were immediately snap-frozen in liquid nitrogen before storage at 280uC before use. Information on clinical variables and patient follow-up were obtained from a prospectively maintained database as well as review of medical records. mRNA Expression – Quantitative Real-time PCR Total RNA was extracted from snap-frozen samples of lung tumor tissue and matched normal tissue by mechanical homog- enization and Trizol. Reverse transcription was performed with random hexamer primers and Superscript II reverse transcriptase (Life Technologies) using 500 ng of total RNA according to the manufacturer’s instructions. One microliter of cDNA was ampli- fied using TaqMan Assays-On-Demand Gremlin gene expression probe (Applied Biosystems, Foster City, CA, assay ID Cf02677902_g1) and GeneAmp 7900 HT Fast sequence detector thermal cycler (Applied Biosystems). Levels of gene expression were determined using the comparative (CT) method with samples analyzed in triplicate. Results were then expressed by a modified Livak formula in which GREM1 expression is normalized to the measured expression of the GAPDH gene [40]. Data are represented as 2-(CT(GREM1)-CT(GAPDH)) for both tumor and Gremlin Overexpression in Lung Adenocarcinoma matched normal specimens. The average delta CT of GREM1 in normal samples was 9.620 and an average tumor delta CT was 5.33, suggesting that GREM1 is overexpressed by more than 19 folds in adenocarcinoma. Immunohistochemistry 5-micron paraffin-embedded tissue sections from matched normal and tumor tissue samples were deparaffinized in xylene and then rehydrated in graded alcohol. Antigen retrieval was achieved by steaming the tissue sections in citrate buffer for 20 minutes (Biogenex ready-to-use antigen retrieval citra). Slides were then washed in PBS, blocked in goat serum (10% diluted in TBS with 1%BSA) for 1 hour, and then incubated in the primary antibody overnight at 4uC. The primary antibody selected was rabbit monoclonal anti-human Gremlin I (ab90670, ABCam) at a 1 to 500 dilution. Following TBST washes, endogenous peroxidase activity was then quenched with 0.3% hydrogen peroxide in TBS. The Invitrogen ABC-DAB (Histostain Plus Broad Spectrum) kit was then used according to the manufac- turer’s protocol. Detection was achieved using a biotinylated anti- goat secondary antibody and DAB chromogen (Invitrogen). The sections were then counterstained with hematoxylin before being mounted with organic media and glass slides. Negative control sections were prepared by the exclusion of the primary antibody to determine the specificity of the staining. Tissue sections were then examined microscopically on an Olympus photomicroscope (Inha, Japan). Plasmids Total RNA was extracted from MRC-5 normal lung fibroblast cells using TRIzol (Invitrogen) and reverse-transcribed using SuperScript One-Step reverse transcription-PCR (RT-PCR). PCR primers were based on human GREM1 cDNA sequences (Gene ID: 26585) as follows: forward: 59-CGGAATTCGATGAGCCG- CACAGCCTACACGGT-39 and reverse: 59-TCGTCTAGAT- TAATCCAAATCGATGGAATAGCAACGACACTGC-39) (EcoRI and Xba I linker sequences are underlined). cDNA encoding human Grem1 was subcloned into the EcoRI and XbaI sites of the vectors p3xFlag-CMC-10 (Sigma). The flag-tagged Grem1 cDNA was released by SpeI and XbaI digest and blunt- ended by Mung-Bean nuclease (NEB) before insertion into SnaBI- linearized pBabe.pur retroviral expression plasmid. Cells and Generation of Stably Transfected cells Human MRC-5 lung fibroblast and BEAS-2B lung epithelial cells were obtained from ATCC (www.atcc.org). MRC-5 and BEAS-2B cells expressing flag-tagged Grem1 were generated by transduction with amphotropic retroviruses (pBabe-pur) in the presence of polybrene (8 mg/ml), using high titer retrovirus supernatants derived from Phoenix packaging cells. After in- fection, MRC-5 and BEAS-2B cells were selected with 2.5 mg/ml puromycin for 6 days and expression of appropriate transgene products confirmed by immunoblotting of cell lysates. To confirm the findings of the colony formation assays, two additional independent lung fibroblast and epithelial cell lines were studied. Human HFL-1 lung fibroblast and NL-20 lung epithelial cells were obtained from ATCC. HFL-1 and NL-20 cells expressing flag-tagged Grem1 were generated by transduction with amphotropic retroviruses (pBabe-pur) in the presence of polybrene (8 mg/ml), using high titer retrovirus supernatants derived from Phoenix packaging cells. After infection, NL-20 and HFL-1 cells were selected with 0.5 mg/ml puromycin for 6 days and expression of appropriate transgene products confirmed by immunoblotting of cell lysates (Fig. S1). PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42264