Figure 1.

NOD1 is highly expressed in human colon cancer tissues, cultured cancer cells and liver metastasis. (A) (i) Immunohistochemical (IHC) staining of human tissue-microarrays (TMA) of early and late stage colorectal adenocarcinoma (CRC) (n = 81 for tumor T, and 86 for normal adjacent tissue NAT, scale bar measures 7 μm). (B) Representative images of tumor core (i, ii) and its paired NAT (iii, iv) from one patient are shown at different magnifications. Scale bar measures 1 mm for (i, iii) and 100 μm for (ii, iv). Insert shows negative control (NEG) which Hematoxylin nuclear counterstaining. (C) Representative images of tumor core (i, ii) and its paired NAT (iii, iv) from another patient are shown at different magnifications. Scale bar measures 1 mm for (i, iii) and 100 μm for (ii, iv). Insert shows negative control (NEG). (D) NOD1 intensity in tumor cores and paired NAT by colon cancer stages. Irrespective of stage, there is higher level of NOD1 in primary colon cancer tumors, as evidenced by the greater number of 3+ staining specimens, compare to their NATs (P < 0.0001). (E) NOD1 expression in cultured HT29 and MC38 cancer cells is identified using flow cytometry with cytosolic staining technique. NOD1 expression (■), secondary antibody control (—), and unstained cells (- - -) are shown. There is significant shift in peak fluorescence for NOD1 protein in all adenocarcinoma cell lines tested (P < 0.05). (F) Immunoblotting is used to confirm the level of NOD1 using whole lysate of the aforementioned cell lines and HEK293 and normal colonic fibroblast tissue CCD18Co, which have low NOD1 expression. The relative NOD1 expression is higher for HT29 and MC38 than HEK293 and CCD18Co