1. Volume 142, Issue 4, Pages 824-833.e7 (April 2012)
Folic Acid Increases Global DNA Methylation and Reduces Inflammation to Prevent Helicobacter-Associated Gastric Cancer in Mice 
Tamas A. Gonda, Young–In Kim, Martha C. Salas, Mary V. Gamble, Wataru Shibata, Sureshkumar Muthupalani, Kyoung–Jin Sohn, Julian A. Abrams, James G. Fox, Timothy C. Wang, Benjamin Tycko 
Gastroenterology 
Volume 142, Issue 4, Pages e7 (April 2012)
DOI: /j.gastro
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2. Figure 1
Global methylation index and methyl donor status in the mouse model of gastric cancer. (A) Results of the [3H]dCTP incorporation assay normalized to the methylation index of wild-type FVB mouse gastric genomic DNA. A significant decrease in the INS-GAS + Helicobacter felis mice at 22 and 28 WPI was seen. (B) Serum total homocysteine (tHCY) levels in WT and INS-GAS mice. A significant difference compared with WT in the INS-GAS + H felis 28 WPI and in the INS-GAS + H felis 22 WPI. (C) Tissue folate content. There is a decrease in tissue folate caused by H felis and a significant reduction in INS-GAS + H felis 28 WPI. Error bars, standard deviation. *P < .05 compared with WT unless otherwise indicated; **P < .001 compared with WT.
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3. Figure 2
Experimental design and FA effects on body weight, serum markers, and Helicobacter felis colonization. (A) Experimental design. Arrow indicates timing of H felis gavage. (B) Body weight of mice at 36 weeks on supplemented and control diet. No significant difference was noted. (C) Serum folate levels. Significant difference noted between control and all FA-supplemented groups. (D) Serum total homocysteine levels. Significant decrease was noted in all FA-supplemented groups compared with controls. (E) Quantitative real-time polymerase chain reaction analysis of H felis DNA from the gastric corpus. Copy number is expressed as the log of copy number of H felis/microgram of stomach DNA. No significant difference was noted among groups. Error bars, standard deviation. *P ≤ .05; **P ≤ N = 47 or 9 to 10/group.
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4. Figure 3
FA supplementation prevents dysplasia and markedly reduces the increase in mucosal thickness and stomach size. (A) Macroscopic appearance of the stomach of INS-GAS H felis-infected mice supplemented with FA compared with mice on no FA control diet. (B) Ratio of stomach wet weight over body weight. Significant reduction is seen in all FA-supplemented groups compared with control (n = 9 to 10/group). (C) Histologic changes in control animals and following FA supplementation started at weaning and at 16 weeks. In the majority of control animals, at least high-grade dysplasia is observed. The majority of animals started on folate at weaning develop at most cellular atypia, indefinite for dysplasia or low-grade dysplasia (typically in cases of folate started at 16 weeks). (D) pH 2.5 PAS/Alcian blue staining. PAS/Alcian blue staining was used to demonstrate gastric (red) or intestinal (blue) mucins or mixed (azure) mucins suggestive of intestinal metaplasia (incomplete or type II). In the no FA control animals, the dysplastic glands show predominantly blue and azure mucin consistent with intestinal metaplasia. However, PAS/Alcian blue staining shows less frequent foci of metaplasia (some azure and foci of blue) in FA-supplemented sections acquired from animals started on FA at weaning. In cases when folate is introduced at a later time point (ie, at 16 weeks) the degree of metaplasia is more evident. Original magnification, ×100; scale bar, 100 μm. (E) Histopathologic changes and degrees of neoplasia in all groups. High-grade dysplasia or carcinoma in situ is only seen in the majority of no FA control samples and in a small minority of supplemented cases. No clear trend is evident based on timing of FA supplementation. *P ≤ .05; **P ≤ .001.
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5. Figure 4
Global hypomethylation is prevented by FA supplementation. (A) Relative content of methylated CpGs based on the 3HdCTP incorporation assay in all FA-supplemented and control gastric corpus DNA. A significant difference was noted at all time points except the latest compared with control. (B) Mouse B1 methylation significant decrease in methylation in the earliest FA supplementation group (n = 9 to 10). (C and D) Anti-5-methylcytosine immunohistochemistry showing an increase in staining intensity between a FA-supplemented (C) gastric corpus and a control (D). *P ≤ .05; **P ≤ N = 47. Original magnification, ×200; scale bar, 50 μm.
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6. Figure 5
Stromal methylation status in FA-supplemented mice. (A–D) Dual immunohistochemistry using anti-5mC (brown) and anti-αSMA (red) in early (A, at weaning) and late (B, 8 weeks) FA-supplemented gastric tissue and in controls (C and D). Majority of αSMA-positive cells (arrows) contained anti-5mC-positive nuclei in the FA-supplemented groups, whereas fewer positive cells were seen in the controls Original magnification, ×400; scale bar, 50 μm. (E) Percent of anti-αSMA- and anti-5mC-positive cells in cases and controls in 8 high-power fields (HPF) per case among all anti-αSMA-positive cells. Significantly greater double positive cells in the FA-supplemented group. *P ≤ .05; **P ≤ (F) Immunofluorescence showed absence of anti-5mC staining (red) in the nuclei of αSMA-positive cells (green) in the control cases, whereas most αSMA-positive cells stained with anti-5mC in the folate-supplemented samples.
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7. Figure 6
Differentially expressed genes and extent of inflammation in gastric tissue of FA-supplemented vs control mice. (A) Sixty-three differentially expressed genes (minimum fold difference of 1.5) between stage-matched folate-supplemented and control tissue. (B) Ontologic categorization of known genes. (C) Mucosal and submucosal T lymphocytes in control compared with FA-supplemented (at 8 weeks) gastric tissue. Anti-CD3 staining shows marked decrease in the number of CD3-positive cells in FA-supplemented samples. Original magnification, ×200; scale bar, 50 μm.
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8. Supplementary Figure 1
Calibration data from mixing experiment. In vitro methylated and unmethylated DNA was used to validate the ability of the cytosince incorproation assay to quantify the average CpG methylation of genomic HpaII sites. This or similar curves were used to calculate the percentage of relative methylation in each experiment. DPM, disintegration per minute.
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9. Supplementary Figure 2
Histologic features of no FA control animals. (A) All animals develop high-grade dysplasia or carcinoma in situ. (B) Submucosal invasion (or herniation) is less commonly seen but develops in 4 of 10 animals. Original magnification, ×400.
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10. Supplementary Figure 3
Decreased proliferation and apoptosis following folic acid supplementation. (A and B) A notable decrease in Ki67 staining can be seen between dysplastic glands in control animals (A) compared with folic acid-supplemented animals (B). In the latter, the majority of Ki67-positive cells is only seen at the base of the glands. (C and D) Caspase 3 staining is seen in the majority gland lumen in control animals (C) but rarely seen in folate-supplemented animals (D). Original magnification, ×100.
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11. Supplementary Figure 4
Comparison of relative methylation, length of time on FA supplementation and dysplasia in FA-supplemented mice. (A) A significant difference in percentage of relative methylation by the Cytosine acceptor assay was noted between animals that received FA early in their life (at weaning and at 0 weeks) vs late (8–16 weeks) (P < .001). (B) No significant difference in percentage of relative methylation by the Cytosine acceptor assay was noted among the animals in the FA group based low (<2.5) and high (≥2.5) dysplasia scores (P = .25).
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12. Supplementary Figure 5
5-Hydroxy-methylyctosine (5HmC) immunostaining in control and folate-supplemented animals (folate initiated 8 weeks). (A) 5HmC staining is relatively preserved in the epithelial cells from no folic acid control animals but is lost in a fraction of spindle-shaped stromal cells. (B) In FA-supplemented animals, a similar pattern of decreased 5HmC staining is seen in the stromal cells. Original magnification, ×400.
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