Figure 1. TLR4 expression is up-regulated in colitis-associated tumors.

A. Expression of TLR4 in normal mucosa and tumors from patients with UC. Samples of tumor tissue (n=4) and corresponding surrounding non-dysplastic colon (n=4) were examined for TLR4 protein by Western blot analysis. Colonic tissues from patients with active ulcerative colitis were also examined (Inflamed) (n=4). 25 μg/lane of protein was loaded per lane. β-actin staining is used as a loading control.

B. Immunofluorescent staining for TLR4 in human colitis-associated cancer. Normal human colon tissue (2 representative examples of 11 patients) (Top panels) and colon cancer in patients with ulcerative colitis (2 representative examples of 15 patients) (middle panels) were stained for TLR4.

C. TLR4 mRNA expression in the AOM-DSS model. Normal colon (n=10), acute phase after 7 days of DSS (n=10), day 56 after injection of AOM (n=5), day 56 of chronic DSS colitis after two cycles of DSS (n=5), non-dysplastic mucosa in AOM+DSS treated mice at day 56 (n=5), and tumor tissues taken from AOM+DSS treated mice at day 56 were analyzed for TLR4 mRNA expression by real-time PCR. P <0.05 for tumor tissue versus non-dysplastic mucosa, non-dysplastic mucosa versus normal colon, and chronic DSS versus normal mucosa.

D. Western blot analysis for TLR4 in murine colon samples. Immunoblots of tissue lysates (25 μg/lane) prepared from colonic samples of tumor versus corresponding surrounding non-dysplastic mucosa (n=4). Graph shows ratios of TLR4 band intensity normalized with β-actin band (ND= Non-dysplastic mucosa, T= Tumor). Mean ratios of non-dysplastic mucosa and tumor are shown at the end (*P<0.0001).

E. Immunofluorescent staining for TLR4 in the mouse CAC model. Serial sections of tumors or surrounding mucosa are stained with H&E for histology and immunofluorescent staining for TLR4. Mesenteric lymph node (MLN) is used as a positive control; mesenteric lymph node from a TLR4−/− mouse is used as a negative control.