-mediated up-regulation of Cox-2 expression. A. Real-time PCR results for mucosal Cox-2 mRNA expression in TLR4-/- mice treated with PGE 2; day 56 in AOM-DSS model. Data are represented as mean (± SD) of relative values of expression (n = 7 PBS group; n = 8 high dose group, PGE 2 200 μg; and n = 6 low dose group, PGE 2 100 μg; n = 5 PGE 2 during DSS treatment, PGE 2 200 μg; *P < 0.05). B. Immunofluorescent staining for Cox-2 in the colon. Representative pictures show Cox-2 positive cells (green - FITC) in lamina propria cells. High dose PGE 2 treated mice show many Cox-2 positive cells in the lamina propria. Low dose PGE 2 treated mice and PBS treated mice have very few Cox-2 positive cells in lamina propria. Double staining demonstrates most Cox-2 positive cells (green - FITC) are also positive for CD68 (red - TRITC) both in the non-tumor area (middle panel) and tumor area (bottom) (serial pictures in same area are shown). Original magnification: 400 ×. Bars indicate 50 μm. C. Western blot analysis of Cox-2 production in murine macrophage cell line RAW246.7 and TLR4-/- peritoneal macrophages. Representative results from three independent experiments. Cells were treated with 10 μM PGE 2 for 18 hours. An equal amount of protein was loaded per lane. The membrane was sequentially probed for Cox-2 and β-actin.