1. Volume 136, Issue 2, Pages 640-651.e1 (February 2009)
Methylation of Protocadherin 10, a Novel Tumor Suppressor, Is Associated With Poor Prognosis in Patients With Gastric Cancer 
Jun Yu, Yuen Y. Cheng, Qian Tao, Kin F. Cheung, Cleo N.Y. Lam, Hua Geng, Lin–Wei Tian, Ying P. Wong, Joanna H.M. Tong, Jian– Ming Ying, Hongchuan Jin, Ka F. To, Francis K.L. Chan, Joseph J.Y. Sung 
Gastroenterology 
Volume 136, Issue 2, Pages e1 (February 2009)
DOI: /j.gastro
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2. Figure 1
(A) Robust mRNA expression of PCDH10 in normal adult tissues and fetal digestive tissues. (B) PCDH10 expression was lost or reduced in all gastric cancer cell lines except YCC11. (C) The mRNA expression of PCDH10 was restored after treatment with demethylation agent 5-Aza. (D) Demethylation was observed in the MKN45 cell line after treatment with demethylation agent 5-Aza and trichostatin (A+T) as examined by bisulfite genomic sequencing. gapdh, glyceraldehyde-3-phosphate dehydrogenase; rt-pcr, reverse-transcription PCR.
Gastroenterology  , e1DOI: ( /j.gastro )
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3. Figure 2
(A) mRNA expression levels of PCDH10 in gastric cancer and their adjacent nontumor tissues as determined by quantitative real-time PCR (n = 12). (B) Combined bisulfite restriction analysis (cobra) of the PCDH10 promoter region in gastric cancer. (B1) BstUI sites and regions for COBRA are indicated. (B2) Representative gel images of the PCDH10 methylation status in gastric cancer, adjacent nontumor, and normal gastric tissue samples. The undigested fragment (upper band) represents the unmethylated DNA (u). The digested fragments (lower bands, indicated by the arrows) represent the methylated DNA (m). The degree of DNA methylation can be estimated by calculating the band intensity of digested fragment (methylated DNA)/undigested fragment (unmethylated DNA). Our result showed that dense methylation was detected in gastric cancer tissues and MKN45 cells, partial methylation was found in adjacent nontumor tissues, and methylation was devoid of normal tissues. The methylation status was indicated in the representative samples.
Gastroenterology  , e1DOI: ( /j.gastro )
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4. Figure 3
The cloned bisulfite sequencing was performed on the PCDH10 genomic region (−328 to +39 bp) relative to the transcription start site (tss). The percentage of methylation on each CpG dinucleotide was calculated by the number of methylated clones in each CpG site divided by the total number of clones in the same CpG site. In vitro methylated DNA (ivd) served as a positive control to ensure complete bisulfite conversion for methylation detection.
Gastroenterology  , e1DOI: ( /j.gastro )
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5. Figure 4
Effect of ectopic PCDH10 expression on tumor growth. (A) Expression of PCDH10 in MKN45 transfected with PCDH10 was confirmed by reverse-transcription PCR. (B) Colony formation assay. Top panel shows the representative dishes of transfection with pcDNA3.1/PCDH10 or empty vector (pcDNA3.1). Quantitative analyses of colony numbers are shown in the lower panel. Values are the mean ± SD of at least 3 independent experiments. **P < (C) Inhibition of colony formation by PCDH10 was confirmed by anchorage-independent growth assay in soft agar. (D) PCDH10 inhibits growth of tumors derived from MKN45 in vivo. (D1) A representative picture of tumor growth in nude mice subcutaneously inoculated with MKN45/vector or MKN45/PCDH10. (D2) Averaged longitudinal tumor growth curves were plotted against days after treatment. Statistical analysis using repeated-measures analysis of variance indicated the MKN45/PCDH10 group showed a significantly slower tumor growth compared with the MKN45/vector group (P < .001) (n = 5/group). (E) Effect of PCDH10 on cell invasion. Gentian violet–stained, invaded MKN45 cells transfected with control (vector) or PCDH10 in a Matrigel invasion assay. The data are means ± SD of 4 separate experiments. *P < .05. (F) Molecular events for induction of cell apoptosis, suppression of cell proliferation, and invasion by PCDH10.
Gastroenterology  , e1DOI: ( /j.gastro )
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6. Figure 5
(A) Kaplan–Meier survival curves show that patients with PCDH10 methylation in adjacent nontumor tissues (methylation) had poorer survival than others (nonmethylation). This difference is statistically significant based on the log-rank test (P < .0001). |, Censored. (B) Kaplan–Meier curves of gastric cancer patients, stratified by methylation status, of 4 different TNM stages. In each panel of patients of the stages I, II, and III, survival was significantly shorter in the methylation group (PCDH10 methylation in adjacent nontumor tissues) than others (nonmethylation). In the stage IV panel, however, overall survival was not significantly different between the methylation and nonmethylation group. The overall survival in years (mean ± SD) for TNM stages I, II, III, and IV is 3.90 ± 2.30, 2.14 ± 0.98, 2.90 ± 2.52, and 0.92 ± 0.86, respectively.
Gastroenterology  , e1DOI: ( /j.gastro )
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