expressed at lower levels in the remaining 8 lines is of interest. There appears to be no correlation between mesothelioma subtypes (our collection included 7 epitheloid, 8 sarcomatoid and 1 biphasic type cell line). Together these data paint a picture of MSLN as a less lineage specific marker that is upregulated in cancer to promote proliferation and migration of tumor cells [51], while LRRN4 and UPK3B are more specific lineage markers that provide no competitive advantage to the tumor so can be lost during tumor progression. With this in mind it may be of interest to consider whether loss of LRRN4 or UPK3B expression correlates with later stage and poorer prognosis, but is beyond the scope of the current manuscript. In conclusion, we identified 311 putative markers of the mesothelial lineage including 48 that were found in pleural, pericardial and peritoneal mesothelium (it is yet to be shown whether they are also expressed in the other rare mesothelial populations at the tunica vaginalis and tunica serosa uteri). We go on to demonstrate that polyclonal antibodies against two of these, LRRN4 and UPK3B react with human mesothelioma tissue localised to the plasma membrane. With this in mind we are currently exploring the development of LRRN4 ELISA based assays for detection of mesothelioma antigens in patient body fluids. Supporting Information Figure S1 Extended In-situ hybridization panels for 10 candi- date biomarkers and GAPDH control. Shows staining pattern References 1. Aoe K, Hiraki A, Fujimoto N, Gemba K, Kishimoto T (2010) The first nationwide survival analysis of Japanese mesothelioma patients from Vital Statistics of Japan. Journal of Clinical Oncology 28: e12007. 2. Ameille J, Ruffie P, Bergeret A (2004) [Asbestos-related occupational cancers]. Rev Prat 54: 1649–1659. 3. Hill ID, Doll R, Knox JF (1966) Mortality among asbestos workers. Proc R Soc Med 59: 59–60. 4. McNulty JC (1962) Malignant pleural mesothelioma in an asbestos worker. Med J Aust 49(2): 953–954. 5. Wagner JC, Sleggs CA, Marchand P (1960) Diffuse pleural mesothelioma and asbestos exposure in the North Western Cape Province. Br J Ind Med 17: 260–271. 6. Armstrong BK, Musk AW, Baker JE, Hunt JM, Newall CC, et al. (1984) Epidemiology of malignant mesothelioma in Western Australia. Med J Aust 141: 86–88. 7. LaDou J, Castleman B, Frank A, Gochfeld M, Greenberg M, et al. (2010) The case for a global ban on asbestos. Environ Health Perspect 118: 897–901. 8. Selikoff IJ, Hammond EC, Seidman H (1980) Latency of asbestos disease among insulation workers in the United States and Canada. Cancer 46: 2736–2740. 9. Sheibani K, Shin SS, Kezirian J, Weiss LM (1991) Ber-EP4 antibody as a discriminant in the differential diagnosis of malignant mesothelioma versus adenocarcinoma. Am J Surg Pathol 15: 779–784. 10. Jasani B, Edwards RE, Thomas ND, Gibbs AR (1985) The use of vimentin antibodies in the diagnosis of malignant mesothelioma. Virchows Arch A Pathol Anat Histopathol 406: 441–448. 11. Benjamin H, Lebanony D, Tabak S, Barabash N, Gibori H, et al. (2009) MicroRNA-based assay for differential diagnosis of mesothelioma. J Clin Oncol, (Meeting Abstracts) 27: e22079-. 12. Beyer HL, Geschwindt RD, Glover CL, Tran L, Hellstrom I, et al. (2007) MESOMARK: a potential test for malignant pleural mesothelioma. Clin Chem 53: 666–672. 13. Li Z-Q, Verch T, Allard WJ (2007) MESOMARK AˆH in vitro diagnostic test for mesothelioma. Expert Opinion on Medical Diagnostics 1: 137–142. 14. Creaney J, Yeoman D, Demelker Y, Segal A, Musk AW, et al. (2008) Comparison of Osteopontin, Megakaryocyte Potentiating Factor, and Mesothe- lin Proteins as Markers in the Serum of Patients with Malignant Mesothelioma. Journal of Thoracic Oncology 3: 851–857: 810.1097/JTO.1090b1013e318- 180477b. 15. Chang K, Pastan I (1996) Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci U S A 93: 136–140. 16. Kojima T, Oh-eda M, Hattori K, Taniguchi Y, Tamura M, et al. (1995) Molecular cloning and expression of megakaryocyte potentiating factor cDNA. J Biol Chem 270: 21984–21990. LRRN4 and UPK3B as Mesothelial Markers across a panel of tissues (Lung, Heart, Liver, Kidney, Spleen, Intestine, Testis and Thymus). (PDF) Figure S2 Peptide sequences used to raise antibodies for LRRN4 and UPK3B. (PDF) Table S1 qRTPCR validation. Includes Illumina probeID, Gene symbol, primer sequences and CT values across a panel of tissues. (XLS) Table S2 In-situ hybridization probes. Includes Symbol, target and primer sequences. (XLS) Table S3 Genes enriched in mesothelium, including B-statistic and fold change. (XLS) Table S4 Gene ontology enrichment analysis of mesothelial enriched gene sets. (XLS) Author Contributions Conceived and designed the experiments: MK-K YH H. Suzuki ARRF. Performed the experiments: MK-K AK YI YO-O MA. Analyzed the data: MK-K ARRF. Contributed reagents/materials/analysis tools: OH MA TK H. Sekihara YN. Wrote the paper: ARRF MK-K. 17. Scholler N, Fu N, Yang Y, Ye Z, Goodman GE, et al. (1999) Soluble member(s) of the mesothelin/megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma. Proc Natl Acad Sci U S A 96: 11531–11536. 18. Eba S, Karube Y, Minowa M, Chida M, Matsumura Y, et al. (2009) [Early malignant pleural mesothelioma; report of a case]. Kyobu Geka 62: 598–601. 19. Du P, Kibbe WA, Lin SM (2008) lumi: a pipeline for processing Illumina microarray. Bioinformatics 24: 1547–1548. 20. Gentleman R, Carey VJ, Huber W, Irizarry RA, Dudoit S, et al. (2005) limma: Linear Models for Microarray Data. In: Dietz K, Samet J, Gail M, Tsiatis A, Krickeberg K, eds. Bioinformatics and Computational Biology Solutions Using R and Bioconductor: Springer New York. pp 397–420. 21. Suzuki H, Okunishi R, Hashizume W, Katayama S, Ninomiya N, et al. (2004) Identification of region-specific transcription factor genes in the adult mouse brain by medium-scale real-time RT-PCR. FEBS Lett 573: 214–218. 22. Jirsova K, Neuwirth A, Kalasova S, Vesela V, Merjava S (2010) Mesothelial proteins are expressed in the human cornea. Exp Eye Res 91: 623–629. 23. Ichikawa M, Okamura-Oho Y, Okunishi R, Kanamori M, Suzuki H, et al. (2005) Expression analysis of genes responsible for serotonin signaling in the brain. Neurobiol Dis 19: 378–385. 24. Usami N, Fukui T, Kondo M, Taniguchi T, Yokoyama T, et al. (2006) Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients. Cancer Sci 97: 387–394. 25. Phelps RM, Johnson BE, Ihde DC, Gazdar AF, Carbone DP, et al. (1996) NCI- Navy Medical Oncology Branch cell line data base. J Cell Biochem Suppl 24: 32–91. 26. Versnel MA, Hoogsteden HC, Hagemeijer A, Bouts MJ, van der Kwast TH, et al. (1989) Characterization of three human malignant mesothelioma cell lines. Cancer Genet Cytogenet 42: 115–128. 27. Versnel M (2002) Mesothelioma. In: Masters J, Palson B, eds. Human Cell Culture: Springer Netherlands. 28. Manning LS, Whitaker D, Murch AR, Garlepp MJ, Davis MR, et al. (1991) Establishment and characterization of five human malignant mesothelioma cell lines derived from pleural effusions. Int J Cancer 47: 285–290. 29. Hagiwara Y, Hamada Y, Kuwahara M, Maeda M, Segawa T, et al. (2008) Establishment of a novel specific ELISA system for rat N- and C-ERC/ mesothelin. Rat ERC/mesothelin in the body fluids of mice bearing mesothelioma. Cancer Sci 99: 666–670. 30. Ishikawa K, Segawa T, Hagiwara Y, Maeda M, Abe M, et al. (2009) Establishment of novel mAb to human ERC/mesothelin useful for study and diagnosis of ERC/mesothelin-expressing cancers. Pathol Int 59: 161–166. 31. Liu CC, Chen WS, Lin CC, Liu HC, Chen HY, et al. (2006) Topology-based cancer classification and related pathway mining using microarray data. Nucleic Acids Res 34: 4069–4080. PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e25391