Supplemental Figure Legends
Supplemental Figure 1. Promoter methylation analysis of the candidate genes in primary colon tissues.
Promoter methylation of candidate genes in CRC cell lines and tissues was confirmed by COBRA after digestion of the gel-extracted PCR product with
BstU 1. Visible COBRA bands were separated on an acrylamide gel. A, COBRA in three
CRC cell lines and 4 normal tissues was performed in parallel to confirm the bisulfitesequencing results of each gene, and was considered as an experimental control for primary tissues.1, HCT116; 2, HT29; 3, DLD-1; 4-7, normal colon tissue samples from patients 23, 33, 34, and 36, respectively. L, 1 Kb plus DNA ladder. B, COBRA in randomly selected matched tumor and normal colon tissues. In contrast to cell lines, hHLP1 and RBP4 exhibited no methylation in 10 primary tumors, indicating that methylation of these genes may have occurred during cancer cell line propagation. The frequency of tumor methylation of DFNA5 (40%, 4/10) and CXX1 (60%, 6/10) was higher than corresponding normal tissues, but CXX1 was also frequently methylated in colon normal tissues (40%, 4/10).
HBA1 was frequently methylated in both normal (50%,
5/10) and tumor tissues (70%, 7/10), although the ladder of PCR products cleaved in tissues was much weaker than those in cell lines. Numbers, primary tissue samples from patients. N, matched normal colon tissues; T, matched CRC tissues.
Supplemental Figure 2. Representative sequencing results of candidate gene promoters. A , DFNA5 was found to be methylated in all cell lines tested (HCT116,
HT29, DLD-1, RKO, SW48, Lovo, and SW480). Representative sequencing results of
DFNA5 ( A ), CXX1 ( B ), HBA1 ( C ), and RBP4 ( D ) in CRC cell lines and primary tissues.
NC, normal colon samples from patients with colon cancer. Total 10 normal and 5 tumor tissues were analyzed for the DFNA5 sequencing. The results of DFNA5 in all CRC cell lines except HCT116 and all five CRC tissues tested were similar to the results in HT29;
No overlap of G (methylated) and A (unmethylated) peaks were observed in the CpG sites in the DFNA5 promoter region analyzed in this study. The overlap of G and A peaks in a few CpG of the DFNA5 promoter region were observed in HCT116 cells, but most of CpG had only methylated peaks. We did not observe any G peaks in normal tissues.
Primer sequences for bisulfite-sequencing and COBRA are underlined and the transcription start sites (TSS) are italicized. Arrows, all guanines present after sequencing that are complementary to methyl cytosines on the opposite DNA strand.
Supplemental Figure 3. DFNA5 inhibits cell growth both in transiently and stably transfected cells. A , DFNA5 expression level in stably expressing HCT116 cells were analyzed by RT-PCR. Data shows 6 single clones and a pooled clone that expressed relatively high level of DFNA5 among total 16 selected clones (data now shown). Clone
#6 was selected for further study. HCT116 cells were transiently (1 X 10 4 cells/well, B ) or stably (200 cells/well, C ) transfected with DFNA5 or empty vector, and the colony focus assays were performed after incubation for two weeks. Colonies were counted and photographed under a microscope.