Zhang et al. BMC Cancer 2011, 11:32 http://www.biomedcentral.com/1471-2407/11/32 Additional material Additional File 1: Figure S1: Specificity of rabbit polyclonal anti- RRIG1 antibody for immunohistochemistry. A, Western blot. RRIG1 protein was first in vitro translated with pGEM and pcDNA3.1 vectors- carrying RRIG1 open-reading frame cDNA and an in vitro protein translation kit. The samples from the in vitro translation were subjected to western blot analysis of RRIG1 expression using our polyclonal anti- RRIG1 antibody. NC, negative control. B, Western blot. Esophageal cancer TE-8S22 and prostate cancer PC3 cells were grown on monolayer for 3 days and total cellular protein was extracted and subjected to western blot with the polyclonal anti-RRIG1 antibody. The vector controlled in vitro translational sample was used for negative control. NC, negative control. NS, non-specific. C, Immunohistochemistry. RRIG-1-negative esophageal cancer cells TE-8 and RRIG-1 stably transfected TE-8-RRIG1 cells were grown in organotypic cultures for 14 days and the 3-D cell layers were subjected to tissue processing and immunohistochemistry with the polyclonal anti-RRIG1 antibody. D, Expression of RRIG1 mRNA and protein in breast tissues using in situ hybridization (ISH, see ref. 5 for the detailed methodology) and immunohistochemistry (IHC). Additional Method Organotypic culture. Esophageal cancer cell lines TE-8 and TE- 8-RRIG1 were seeded on to collagen gels prepared by using type I rat- tail collagen (Collaborative Biomedical Products, Bedford, MA), and incubated overnight until the cells will be confluent. The gels were then elevated to the air-liquid interface by placing them onto a surgical stainless steel mesh platform. DMEM was added until it reached the undersurface of the collagen gel but not cover the cell layer. The cultures were incubated at 37°C for 2 weeks after elevation to the air- medium interface. In the end of the experiments, the cell discs were fixed in 4% paraformaldehyde and embedded in paraffin following by sectioning and staining. Acknowledgements We thank the Department of Scientific Publication at MD Anderson Cancer Center for editing the manuscript. This study was supported in part by National Cancer Institute grants R01 CA 117895 and R01 CA078480 and an Institutional Research Grant from MD Anderson Cancer Center as well as grants from The Science Foundations of Education Department of Anhui Province, China (KJ2007B195) and The Youth Foundations of Education Department of Anhui Province, China (2007jq1075). Author details 1Department of Clinical Cancer Prevention, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. 2Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. 3Department of Pathology, Anhui Medical University, Hefei, Anhui, China. Authors’ contributions GZ performed most of the experiments. AB helped in research design and immunohistochemistry. BG performed experiments for T47D cells. ZF provided breast cancer cell lines and contributed to the research design, critical comments, and writing. PHB provided financial support and contributed to the research design. XCX participated in the overall research design, data collection, and writing. All authors read and approved the final Manuscript. Competing interests The authors declare that they have no competing interests. Received: 18 October 2010 Accepted: 25 January 2011 Published: 25 January 2011 References 1. 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