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1 Supplementary Material Fig. S1. Gpa33 -/- mice do not express Gpa33 mrna or GPA33 protein. (A) The Gpa33 null allele contains a premature translational stop codon (STOP) in exon 2, and almost all of the coding sequence is replaced with an IRES and -geo gene. (B) Gpa33 mrna expression in epithelial cells extracted from the small and large intestine of 10 week old Gpa33 -/- mice is negligible, whereas WT mice exhibit robust expression. Data were normalised to Hprt1 expression. (C) Representative GPA33 immunohistochemistry in the small and large intestine reveals strong cell surface expression of GPA33 in intestinal epithelial cells of 10 week old WT mice; this is completely absent in Gpa33 -/- mice. Mean ± SEM, n = 3, ** P <.01, scale bars 50 m.
2 Fig. S2. Flow cytometry analysis of haematopoietic cells within the colon LP and spleen. Single cell suspensions prepared from colon lamina propria (LP) and spleen were analysed by flow cytometry for myeloid and lymphoid populations. Absolute cell numbers for whole colon LP or spleen were calculated using relative cell populations and viable single cell counts. (A) B-cell (Epcam - CD45 + CD11b - TCR - B220 + ) and T-cell (Epcam - CD45 + CD11b - TCR + B220 + and CD4 + or CD8 + ) populations were unchanged in the colon LP of Gpa33 -/- and WT mice. (B) Dendritic cells (DCs; Epcam - CD45 + CD11c + F4/80 - MHC-II + ), activated DCs (Epcam - CD45 + CD11c + F4/80 - MHC-II +, CD86 + ) and macrophages (Epcam- CD45 + CD11c + F4/80 + ) populations were unchanged in the spleen of Gpa33 -/- and WT mice. (C) CD8 + T cell (Epcam - CD45 + CD11b - TCR + B220 - CD8 + ) numbers were slightly elevated in the spleen of Gpa33 -/- and WT mice, whereas B-cell (Epcam - CD45 + CD11b - TCR - B220 + ) and CD4 + T-cell (Epcam - CD45 + CD11b - TCR + B220 - and CD4 + ) numbers were unchanged. Mean ± SEM, n = 6, * P <.05.
3 Fig. S3. Gpa33 is not expressed in haematopoietic populations. Expression of Gpa33 in 39 haematopoietic populations within the bone marrow, spleen and thymus was analysed using the Gene Expressions Commons (GEXC) Mouse Haematopoiesis Model (dataset GSE34723 (Seita et al., 2012)). This model separated haematopoietic cell populations identified by expression of unique combinations of surface markers with flow cytometry cell sorting. Gene expression was analysed for each cell population on the Affymetrix Mouse Genome Array (Affymetrix, Santa Clara, CA, USA) microarray platform. Gene expression activity < 0 indicates that Gpa33 is not expressed in any haematopoietic cell populations. Gpa33 expression activity was analysed using two independent probesets; (A) probeset 1 and (B) probeset 2. (C) Abbreviations used for cell populations in (A, B). Mean ± SEM, n = 3.
4 Fig. S4. Both Gpa33 -/- and WT mice exhibit activation of the WNT pathway in colitis associated tumours. (A) Representative H&E and -catenin immunohistochemistry of CAC tumours from Gpa33 -/- and WT mice reveals elevated -catenin expression within tumours compared to normal epithelium. (B) Relative mrna expression of WNT pathway target genes CD44 and Lgr5 is increased in CAC tumours compared to normal colonic epithelium. Data were normalized to Hprt1 expression. Mean ± SEM, n = 5-9 for (A, B) and 4-8 for (D), * P <.05, ** P <.01, scale bar 100 m.
5 Fig. S5. EPCAM and FAP expression in the inflamed bowel of IBD patients. Relative EPCAM and FAP expression in normal (N), uninflamed (U) and inflamed (I) bowel of IBD patients represented as whisker and box plots. (A) EPCAM expression is not significantly different in inflamed compared to uninflamed bowel from Crohn s disease patients (uninflamed n = 18, inflamed n = 8; dataset GDS3119) (Olsen et al., 2009) or (B) in inflamed compared to normal and uninflamed bowel from ulcerative colitis patients (normal n = 22, uninflamed n = 15, inflamed n = 19; dataset GDS3268) (Noble et al., 2008). (C) FAP expression is not significantly different in inflamed compared to uninflamed bowel from Crohn s disease patients, but is significantly higher in the inflamed compared to the normal and uninflamed tissue from the ulcerative colitis patients (D) * P <.05, ** P <.005. Relative gene expression values were normalised using (A) Human Genome U133 Plus 2.0 Array Normalisation Controls (Affymetrix) and (B) Stratagene Universal Human Reference (Stratagene).
6 Table S1. Histological colitis scoring system The total colitis score comprises epithelial damage and inflammation scores for the proximal, mid and distal colon. Colitis criteria Scoring Tissue compartments Epithelial damage 0 normal Epithelium 1 hyperplasia 2 <50% crypt loss 3 >50% crypt loss 4 100% crypt loss 5 ulceration Presence of inflammatory 0 none Mucosa cells 1 mild Submucosa 2 moderate 3 severe Muscle References Johnstone, C. N., Tebbutt, N. C., Abud, H. E., White, S. J., Stenvers, K. L., Hall, N. E., Cody, S. H., Whitehead, R. H., Catimel, B., Nice, E. C. et al. (2000). Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 279, G Seita, J., Sahoo, D., Rossi, D. J., Bhattacharya, D., Serwold, T., Inlay, M. A., Ehrlich, L. I., Fathman, J. W., Dill, D. L. and Weissman, I. L. (2012). Gene Expression Commons: an open platform for absolute gene expression profiling. PLoS One 7, e40321.
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