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Volume 138, Issue 1, Pages 98-107.e4 (January 2010) Neuropeptide S Receptor Induces Neuropeptide Expression and Associates With Intermediate Phenotypes of Functional Gastrointestinal Disorders Michael Camilleri, Paula Carlson, Alan R. Zinsmeister, Sanna McKinzie, Irene Busciglio, Duane Burton, Marco Zucchelli, Mauro D'Amato Gastroenterology Volume 138, Issue 1, Pages 98-107.e4 (January 2010) DOI: 10.1053/j.gastro.2009.08.051 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 NPSR1 is expressed in intestinal enteroendocrine cells. Immunohistochemistry stainings performed with an affinity purified NPSR1-specific antibody (HPA007489) on human normal intestinal tissue, reported in the Human Protein Atlas portal (http://www.proteinatlas.org). Full-size images and additional information are publicly available at the Swedish Human Proteome Resource program's Web site. Gastroenterology 2010 138, 98-107.e4DOI: (10.1053/j.gastro.2009.08.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Effect of NPS on neuropeptide expression. HEK293 cells were transfected in duplicate experiments with NPSR1, complementary DNA, or empty vector (ctrl) and stimulated with 1 μM NPS for 6 hours. Messenger RNA levels for each gene were measured by quantitative real-time polymerase chain reaction and are expressed as relative fold induction (+standard deviation), compared to control cells. CALCA, calcitonin-related polypeptide α; CCK, cholecystokinin; GHRL, ghrelin; MLN, motilin; nd, not detected; PYY, peptide YY; PPY, pancreatic polypeptide; SST, somatostatin; VIP, vasoactive intestinal peptide. Gastroenterology 2010 138, 98-107.e4DOI: (10.1053/j.gastro.2009.08.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 LD map and characteristics of the studied NPSR1, SNPs. Left: LD and haplotype block structure (LD structure) obtained from Haploview 4 analysis of genotyping data. The numbers in each box correspond to LD coefficient D' between respective SNPs (only values for LD <100% are reported). Center: SNPs (marker) are listed with alleles at each locus (SNP, minor allele in lower case) and minor allele frequency in controls (MAF ctrl). Right: position of each SNP within NPSR1 coding region (gene region), and corresponding effect on messenger RNA (mRNA). Gastroenterology 2010 138, 98-107.e4DOI: (10.1053/j.gastro.2009.08.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Electrophoretic mobility shift assay analysis of the NPSR1 rs1379928. Nuclear proteins from SH-SY5Y (neuroblastoma), Colo205(colon adenocarcinoma), and HEK293(embryonic kidney epithelial) cells were extracted and mixed with radiolabeled oligonucleotide duplexes corresponding to the 2 alleles at the rs1379928 SNP as indicated. Binding reactions were then run on a 6% polyacrylamide gel, which was autoradiographed after electrophoresis to reveal the presence of DNA protein complexes. No discernible differences between alleles were detected in either cell line, although nuclear proteins from SH-SY5Y and HEK293 gave rise to a shifted band that was absent in Colo205 cells. Gastroenterology 2010 138, 98-107.e4DOI: (10.1053/j.gastro.2009.08.051) Copyright © 2010 AGA Institute Terms and Conditions