Volume 137, Issue 1, Pages 176-187 (July 2009) Frequent Inactivation of Axon Guidance Molecule RGMA in Human Colon Cancer Through Genetic and Epigenetic Mechanisms  Vivian S.W. Li, Siu Tsan Yuen, Tsun Leung Chan, Helen H.N. Yan, Wai Lun Law, Bonnie H.Y. Yeung, Annie S.Y. Chan, Wai Yin Tsui, Samuel So, Xin Chen, Suet Yi Leung  Gastroenterology  Volume 137, Issue 1, Pages 176-187 (July 2009) DOI: 10.1053/j.gastro.2009.03.005 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 (A) Genomic structures of the 2 RGMA transcripts. Black bars are the RT-PCR primers; gray bars, TaqMan primers and probe. (B) Map of RGMA promoters, positions of CpG sites (vertical bars) and primers for methylation studies. 42bp indicates 42-bp deletion polymorphism. Gastroenterology 2009 137, 176-187DOI: (10.1053/j.gastro.2009.03.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 (A) RGMA and NEO1 expression by microarray. Data are normalized against the mean of normal mucosae for CRCs and adenomas, and mean centered for colon top versus bottom crypts. Red and green colors indicate above and below mean expression according to the color scale bar; gray color, missing data. (B) Differential expression of RGMA and NEO1 in colon crypts and CRCs by qRT-PCR. The expression level is normalized against the mean of colon bottom crypt. *P < .05; **P < .001. (C) Reduced protein levels of RGMA and NEO1 in CRCs (T) compared with their corresponding normal colon mucosae (N). Gastroenterology 2009 137, 176-187DOI: (10.1053/j.gastro.2009.03.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 (A) RGMA and NEO1 expression by RT-PCR (top) and qRT-PCR in relation to promoter methylation by MSP and pyrosequencing in 13 colon cell lines. NC indicates normal colon mucosa. (B) Methylation status in 6 CpG sites by pyrosequencing in the RGMA promoter. Re-expression of the RGMA transcripts (C) and demethylation of the RGMA promoter in DLD1 and SW48 after 5aza-dC treatment by both pyrosequencing (C) and MSP (D). The # indicates heterozygous for 42-bp polymorphism. Gastroenterology 2009 137, 176-187DOI: (10.1053/j.gastro.2009.03.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 (A) Schematic summary of pyrosequencing results in 61 CRC/normal colon pairs and 11 adenomas. The - indicates the case was excluded because of incomplete bisulfite conversion; dotted line, the methylation cutoff for scoring tumors as positive for methylation (mean > 10%). (B) Mean methylation percentage of the RGMA promoter as measured by pyrosequencing in colon tissues and cell lines. **P < .001 (C) Schematic summary of LOH results from the 7 microsatellite markers on chromosome 15q. Numbers in the brackets represent base pair positions away from the RGMA gene. (D) The bar chart summarizes distribution of the different RGMA-inactivating events among the 61 CRCs. Gastroenterology 2009 137, 176-187DOI: (10.1053/j.gastro.2009.03.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 (A) RGMA expression was confirmed by RT-PCR (top) and Western blot (bottom) after transfection. DLD1/Mock3 and DLD1/RGMA16 indicate cloned stable transfectants; pooled, pooled stable transfectants. Expression of RGMA inhibits colony formation (B), wound-healing ability (C), migration, and invasion by transwell assays (D). Values represent mean from triplicate wells ± 1 SD and normalized to 1 relative to Mock. Gastroenterology 2009 137, 176-187DOI: (10.1053/j.gastro.2009.03.005) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Expression of RGMA enhances apoptosis after camptothecin (CPT) treatment in DLD1 stable clone (A and B) and LOVO transient transfectants (C and D). Apoptosis was measured by flow cytometry after propidium iodide (PI) staining and also counting of apoptotic cells after DAPI nuclei staining (white arrows). Values represent mean from triplicate experiments ± 1 SD. Gastroenterology 2009 137, 176-187DOI: (10.1053/j.gastro.2009.03.005) Copyright © 2009 AGA Institute Terms and Conditions