Volume 118, Issue 2, Pages 337-345 (February 2000) Cyclooxygenase 2 expression is increased in the stroma of colon carcinomas from IL– 10−/− mice  Rebecca L. Shattuck-Brandt, Gary W. Varilek, Aramandla Radhika, Fajun Yang, M.Kay Washington, Raymond N. DuBois  Gastroenterology  Volume 118, Issue 2, Pages 337-345 (February 2000) DOI: 10.1016/S0016-5085(00)70216-2 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 COX-2 mRNA expression is increased in IL-10−/− tumors. (A) Proximal (PC) and distal (DC) segments of the colon from IL-10−/− mice were isolated and analyzed for COX-1, COX-2, and β-actin mRNA by RT-PCR. (B) Individual cecum tumors (T), normal adjacent cecum tissue (N), and an inflamed region of the colon containing no visible tumors from IL-10−/− mice were isolated and analyzed for COX-2, COX-1, and β-actin mRNA expression. Isolated tissue was homogenized in TRI-reagent, and total RNA was isolated by the recommended protocol. One microgram of the isolated RNA was used as a template for cDNA generation by using the enzyme Superscript (+) (Life Technologies, Grand Island, NY). Matched reactions were performed without Superscript (−) to determine the levels of genomic contamination (Genomic). The cDNAs were amplified by multiplex PCR using primers specific for COX-2 and β-actin or COX-1 and visualized by 1.8% agarose gel electrophoresis. RNA isolated from the colon and small intestine of a C57BL/6J wild-type mouse was used as a negative control for COX-2 expression (data not shown). Gastroenterology 2000 118, 337-345DOI: (10.1016/S0016-5085(00)70216-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 COX-2 protein expression is increased in IL-10−/− tumors. Tissue isolated from either individual colon (T) or cecum (cT) tumors or normal adjacent tissue (N) was homogenized in RIPA buffer, and 50 μg of total soluble protein was separated by SDS-PAGE and then analyzed for (A and C) COX-2 or (B) COX-1 expression by Western blot analysis. For both COX-1 and COX-2, proteins were separated by 7.5% SDS-PAGE, and Western blot analysis was performed with polyclonal antibodies to murine COX-1 or COX-2. The immune complexes were detected by using horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence. Protein extracted from the colon of a C57BL/6J wild-type mouse was used as negative control (WT). In addition, an inflamed region of the colon was analyzed (IB). The number of each IL-10−/− mouse is indicated above the samples. Gastroenterology 2000 118, 337-345DOI: (10.1016/S0016-5085(00)70216-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 COX-2 is localized to tumors from IL-10−/− mice. COX-2 immunohistochemical analysis and in situ hybridization were performed on formalin-fixed, paraffin-embedded sections from (A–D) colon tumors or (E–H) cecum tumors of IL-10−/− mice. (A and E) Immunolocalization of COX-2 protein was performed with a rabbit polyclonal antibody to murine COX-2. (B and F) Specificity was determined by preincubating the antibody with the antigenic peptide. The immunoreactive complex was visualized by using a biotinylated secondary antibody and a Vectastain ABC kit. True Blue was used as the substrate and Contrast Red as the counterstain. (C and G) For in situ hybridization, sense (data not shown) and antisense COX-2 riboprobes were labeled with [35S]uridine triphosphate. Sections were hybridized with the riboprobes and washed as described in Materials and Methods. The detected COX-2 mRNA was visualized by autoradiographic emulsion, and the sections were counterstained with Meyer's hematoxylin. Images were taken by using bright-field illumination with a 20× objective. (D and H) Sections stained with H&E. The photomicrographs are representative of results obtained from 6 individual mice. Gastroenterology 2000 118, 337-345DOI: (10.1016/S0016-5085(00)70216-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 COX-2 and α–smooth muscle actin are colocalized within tumors from IL-10−/−, Min, and AOM-treated mice. COX-2 and α–smooth muscle actin immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded sections of tumors from (A and B) IL-10−/−, (C and D) AOM-treated, and (E and F) Min mice. Immunolocalization of COX-2 protein was performed with a rabbit polyclonal antibody to murine COX-2 (A, C, C2, E, and E2), and specificity was determined by preincubating the antibody with the antigenic peptide (data not shown). The immunoreactive complex was visualized by using a biotinylated secondary antibody and a Vectastain ABC kit. True Blue was used as the substrate and Contrast Red as the counterstain. (B, D, and F) α–Smooth muscle actin was detected using clone 1A4 from Sigma with an all-purpose secondary antibody from Ventana. The immune complex was detected with diaminobenzoic acid as the substrate and counterstained with hematoxylin. Images were taken by using bright-field illumination with a 20× (E, F), 40× (A–D), or 100× with oil (C2, E2) objective. The asterisk indicates regions of colocalization. Gastroenterology 2000 118, 337-345DOI: (10.1016/S0016-5085(00)70216-2) Copyright © 2000 American Gastroenterological Association Terms and Conditions