A blinded test was performed for gene methylation analysis in stool DNA collected from patients with or without colon cancer. SFRP1 was included to compare methylation frequency of a known cancer-specific methylated gene. Sensitivity/specificity based on optimal cut-offs calculated from ROC analysis is shown in Table 4.

Strong expression of OSMR was detected in all non-malignant normal tissues (NN) and adjacent normal colon mucosa (PN). In contrast, OSMR was rarely detected in the neoplastic cells from colon cancer patients. Tumor grades are indicated.

A, Methylation status of each gene was examined in CRC cell lines and tissues by C-MSP after PCR with methylation-specific primers. PCR products were run on 4% agarose gels pre-stained with ethidium bromide. In vitro methylated, bisulfite-treated human normal lymphocyte DNA (NL) was used as a positive control for PCR, and distilled water (DW) was used as a negative PCR control. β-actin was used to confirm integrity of DNA. SLC39A4 and SLC9A3R1 were methylated both in cell lines and tissues, but GANAB, PLAU and UBE3A were not. The other 5 genes were methylated in one of three cells lines tested, and only methylated in CRC tissues (PT). PN, paired normal colon tissues from colon cancer patients; PT, paired CRC; NN, normal colon epithelium from non-cancer patients. C-MSP results of OSMR in CRC cell lines and tissues were shown in a previous report [12]. B, Scatter plots of methylation values of six candidate genes in tissues and cell lines (CL). The overall methylation values (TaqMan methylation values, TaqMeth V) and the optimal specificity/sensitivity at optimal cut-offs calculated from ROC analysis are shown in Table 3. Arrows indicate the optimal cut-off values for each gene. No cases of NN displayed TaqMeth V over the optimal cut-offs for B4GALT1 and OSMR. HEK293, a non-tumorigenic cell line, harbored a high level of B4GALT1 methylation (TaqMeth V, 101.12), but OSMR methylation was not detected in the cell line (TaqMeth V, 0.00). *, Samples with a ratio equal to zero could not be plotted correctly on a log scale, so are presented here as 0.1. All assays were performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results in triplicate. TaqMeth V is described in Materials and Methods. C, Quantitative methylation levels of B4GALT1 and OSMR in primary colon tissues. Scatter plot of B4GALT1 and OSMR promoter methylation. Arrows indicate optimal cut-off values for each gene (3.877 for B4GALT1 and 22.01 for OSMR).

A, Expression of B4GALT1 and OSMR in cell lines was examined by RT-PCR analysis. Methylation of B4GALT1 in the cell lines was examined by C-MSP or TaqMan-MSP. β-actin was used as a loading control. M, methylation; U, unmethylation. B and C, Real-time RT-PCR was performed in five pairs (a – e) of normal (PN) and tumor cDNA prepared from CRC patients (PT) (left). Expression of B4GALT1 (B) and OSMR (C) were also compared between patients with colon cancer (T) or without cancer (NN) (right). Relative expression (Fold) was calculated by comparing the ratios of mRNA expression of B4GALT1 and OSMR to an internal control gene, GAPDH. Experiments were done in duplicate, and values indicate means±SD. *, P<0.05. Experiments were done in duplicate, and values indicate means±SD. *, P<0.05.

A, Analysis of OSMR promoter activity by luciferase reporter assay in OSMR-positive (HCT116 and HEK293) and - negative (SW480) cells. The promoter construct was pre-treated with or without SssI methylase for 8 hrs before transfection. The highest activity was detected in HEK293 cells. Data are expressed as fold increase over pGL3-basic activity. Experiments were done in triplicate, and values indicate means±SD. Mean values are presented. B, An siRNA pool targeting OSMR and non-targeting control were transiently transfected into HCT116, and cells were treated with rhOSM (50 ng/ml) (left). HCT116 cells were treated with or without Stat3 inhibitor peptide (Stat3 Inh, 100 μM) acting as a highly selective, potent blocker of Stat3 activation (right). Cell growth was determined by MTT assay. Data are expressed as absorbance at 570 nm. Experiments were done in triplicate, and values indicate means±SD. *, P<0.05. C, 5-Aza-dC (5 μM) was pre-treated for 48 hrs in SW480 and DLD-1 cells, and then co-treated with rhOSM for 48 hrs. Cell growth was determined by MTT assay. *, 5-Aza-dC-treated cells compared to untreated control (P<0.05); #, 5-Aza-dC/rhOSM- treated cells compared to 5-Aza-dC treatment alone (P<0.05). D, The phosphorylation of Stat3 and Erk in response to rhOSM (50 ng/ml) was analyzed by Western blotting in HCT116 and SW480 cell lines. rhOSM increased phosphorylated Stat3 and Erk in HCT116 cells but not in SW480 cells (which lack LIFR-see E below). Equal protein loading was monitored by total Stat3, Erk and β-actin evaluation in the samples. Re-activation of OSMR by 5-Aza-dC treatment was determined on SW480 and DLD-1 cells by flow cytometry (Figure S6). E. Expression of LIFR and gp130 was examined in CRC cell lines by RT-PCR. LIFR was expressed in HCT116 cells but silenced in most other CRC cell lines tested. gp130, the OSMR heterodimer, was ubiquitously expressed in all cell lines tested. Promoter methylation status of these two latter genes was not examined.