path4173-sup-0001-AppendixS1.docWord document21KAppendix S1. Supplementary materials and methods
path4173-sup-0002-FigureS1.tifTIFF image137KmRNA expression and promoter DNA methylation of BEX1 and LDOC1 in OSCC cell lines. Data represent the mean values from triplicate experiments (mean ± SD). CGHNK6 is an immortalized untransformed cell line derived from oral keratinocytes
path4173-sup-0003-FigureS2.tifTIFF image189KMethylation status of BEX1 and LDOC1 in OSCC samples. (A) The methylation index (MI) was defined and measured as described in Materials and methods. Each box-plot represents the MI of BEX1 (left) and LDOC1 (right) for the tumour (OSCC) and paired non-tumour (non-OSCC) samples; p values were calculated using the paired t-test. (B) Frequencies of BEX1 and LDOC1 promoter methylation in OSCC tumour and non-OSCC tissue groups. According to the value of MI measured from CGHNK6 cells, 'methylated' was defined as MI ≥ 10. (C) Frequencies of BEX1 and/or LDOC1 promoter methylation in the OSCC primary tumours
path4173-sup-0004-FigureS3.tifTIFF image1470KBisulphite sequence analysis of CpG islands in the promoter region of the BEX1 and LDOC1 genes in OSCC cell lines and paired tissue samples. Representative results of cloning and sequencing are shown. Each square represents a single CpG site, with open squares representing unmethylated CpG sites and filled squares representing methylated CpG sites. Each row of squares represents one cloned PCR sequence across the gene promoter
path4173-sup-0005-FigureS4.tifTIFF image1017KExpressions of BEX1 and LDOC1 in OSCC transfectants and mouse tumours. (A) The lentivirus infected stable cell pools derived from SCC-15 were used for colony-forming assay and xenograft tumourigenicity assay. (B) Transiently transfected OC3 cells were used for NF-κB reporter assay. The mRNA expression of BEX1 and LDOC1 were determined by RT–PCR. DNA fragments amplified from the indicated plasmid DNA were used as positive controls. (C) Quantitative RT–PCR for BEX1 and LDOC1 with RNA samples obtained from murine xenograft tumours growing from BEX1-expressing, LDOC1-expressing and vector control groups
path4173-sup-0006-FigureS5.tifTIFF image128KAltered methylation status may be caused by exposure to alcohol, betel quid or cigarette smoke. (A) Levels of DNMT3B mRNA expression in the tumour (OSCC) and paired non-tumour (non-OSCC) tissues of ABC-associated OSCC; clinical array data in this study (GSE3799). (B) CAL27 OSCC cells were cultured in growth medium containing mainstream cigarette smoke condensates (CSC, 0.1 µg/ml) for 3 months. Methylation index (MI) were determined by qMSP with DMSO-treated cells as control. (C) Comparison of the average MI of BEX1 and LDOC1 in the tumour (OSCC), the adjacent non-tumour epithelia (non-OSCC) of ABC patients, an immortalized untransformed oral epithelial cell (CGHNK6) and human primary oral keratinocytes (HOK)

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