<p><b>A)</b> Bioinformatics analysis of the CpG island of <i>KIAA1199</i>: Two major subregions within the CpG island were examined by MSP method for MCF-7 and MDA-MB-231 cells. F: forward primers and R: reverse primers. <b>B)</b> Pyrosequencing analysis for quantitation of methylation of cytosine residues in the second subregion of the CpG island between MCF-7 and MDA-MB-231 cells: The percentage of methylation for 36 CG pairs between +525 and +1059 was shown individually (Left panel). Average methylation rate was calculated for MCF-7 and MDA-MB-231 cells (p<0.001) (Right panel). <b>C)</b> Effect of 4 days treatment with 5′-azaon <i>KIAA1199</i> expression: Real time RT PCR was performed in MCF-7 and MDA-MB-231 cells treated with 5′-aza using <i>KIAA1199</i> specific primers. Housekeeping genes were used to normalize the gene expression. <b>D)</b> Methylation status of <i>KIAA1199</i> in human breast cancer specimens: Human invasive breast cancer cells as well as normal breast epithelial cells were harvested by LCM and pyrosequencing was performed using bisulfite-treated DNA. The average methylation level in normal cells was found higher than in cancer cells. A pair of normal breast epithelial cells and breast cancer cells from patients A and B was labeled. n: case number. <b>E)</b><i>KIAA1199</i> expression in human breast cancer specimens: Total RNA from micro-dissected human breast cancer cells as well as normal breast epithelial cells was examined by real time RT-PCR using <i>KIAA1199</i> specific primers. Housekeeping genes were used to normalize the gene expression. A pair of normal breast epithelial cells and breast cancer cells from patients A and B was labeled. n: case number. <b>F)</b> Hypomethylation of <i>KIAA1199</i> in invasive human breast cancer specimens: Bisulfite-treated DNA from paired normal and cancer cells of breast cancer specimens harvested by laser microdissection technique was examined by a pyrosequencing approach. Representative methylation profile between the +932 and +1025 was shown for the benign and invasive cells of single patients.</p
