Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay

Rauch et al. 10.1073/pnas.0701059104.

Supporting Information

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Fig. 5. Bisulfite sequencing data of three CpG islands in the HOXA cluster of GM06990 cells.

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Fig. 6. Methylation analysis of several CpG islands in the TBX20 and Engrailed-1 (EN1) genes. Methylated CpG island recovery assay (MIRA)-enriched fractions from normal human bronchial epithelial (NHBE) cells and A549 lung cancer cells were mixed and hybridized onto Agilent CpG island arrays. The red columns indicate the signal ratio (A/N) between MIRA-enriched A549 DNA (A) versus MIRA-enriched NHBE DNA (N) and represent the relative methylation level. Several separate CpG islands were moderately to strongly hypermethylated near the TBX20 and EN1 genes.

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Fig. 7. Methylation profile analysis of HOX6 and HOX7 paralogous genes. MIRA-enriched fractions from NHBE and A549 cells were mixed and hybridized onto Agilent CpG island arrays. Each probe on the Agilent array is shown by a gray box. The red columns above these small boxes indicate the signal ratio (A/N) between MIRA-enriched A549 DNA (A) versus MIRA-enriched NHBE DNA (N) and represent the relative methylation level. The green horizontal bars show the CpG islands associated with these gene loci. Combined bisulfite restriction analysis (COBRA) assays confirming the MIRA-based data were conducted for the regions indicated in pink (bottom). -, control digestion with no BstUI; +, BstUI-digested samples.

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Fig. 8. DNA methylation profiles of chromosomes 2, 7, 12, and 17 in a primary lung adenocarcinoma versus matching normal lung tissue. MIRA-enriched fractions from a stage 1 adenocarcinoma and adjacent normal lung were mixed and hybridized onto Agilent CpG island arrays. The ratio (fold difference between tumor and normal tissue) plotted indicates individual probe signals (blue diamonds) for hypermethylated CpG islands in the tumor. The numbers indicate selected areas of hypermethylated CpG island clusters including all four HOX gene clusters.

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Fig. 9. DNA methylation profiles of chromosomes 2, 7, 12, and 17 in a primary lung squamous cell carcinoma versus matching normal lung tissue. MIRA-enriched fractions from a stage 1 squamous cell carcinoma and adjacent normal lung were mixed and hybridized onto Agilent CpG island arrays. The ratio (fold difference between tumor and normal tissue) plotted indicates individual probe signals (blue diamonds) for hypermethylated CpG islands in the tumor. The numbers indicate selected areas of hypermethylated CpG island clusters including the HOXA and HOXD gene clusters.

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Fig. 10. Methylation of the HOXA7 and HOXA9 CpG islands in lung squamous cell carcinoma samples. Methylation analysis of the HOXA7 (A7) and HOXA9 (A9) CpG islands was conducted by BstUI COBRA assays. T, tumor; N, normal tissues; -, control digestion with no BstUI; +, BstUI-digested samples. Samples 1, 4, 7, 8, 11, 15, and 19-22 were stage 1 tumors and are circled.