Figure 5

Transgenic cLys-Cox-2 x Apc ^Min/+ mouse colonic tumours have increased tumour-associated angiogenesis and nuclear β-catenin localisation in dysplastic epithelial cells. (A) Immunohistochemistry for mouse CD31 demonstrating capillary microvessels in a non-transgenic Apc ^Min/+ mouse colonic tumour. Size bar = 50 μm. (B) CD31-positive endothelial cells in microvessels in a transgenic cLys-Cox-2 x Apc ^Min/+ mouse colonic tumour. Size bar = 50 μm. (C) Microvessel density (number of CD31-positive foci per ‘hotspot’ 100 x microscopic field) in Apc ^Min/+ mouse colonic tumours. Bars represent the mean and SEM of the MVD for non-transgenic Apc ^Min/+ mouse tumours (n = 8) and transgenic cLys-Cox-2 x Apc ^Min/+ mouse tumours (n = 6). *P = 0.25; Student’s t test). (D) Immunohistochemistry for β-catenin on a transgenic cLys-Cox-2 x Apc ^Min/+ mouse colonic tumour demonstrating strong nuclear and cytoplasmic β-catenin immunoreactivity (score 4) in dysplastic epithelial cells (black arrows) compared with the membranous distribution of β-catenin and absence of nuclear staining in neighbouring non-neoplastic epithelium (white arrow). Size bar = 50 μm. (E) Immunohistochemistry for β-catenin on a non-transgenic Apc ^Min/+ mouse colonic tumour demonstrating patchy, weak nuclear β-catenin staining (score 1) in dysplastic epithelial cells. Size bar = 50 μm. (F) Nuclear β-catenin scores of non-transgenic Apc ^Min/+ (n = 7) and transgenic cLys-Cox-2 x Apc ^Min/+ (n = 9) mouse colonic tumours. P = 0.03, Mann-Whitney U test). (G) Western blot analysis of β-catenin protein content of rat IEC-6 epithelial cells. Cells were cultured for 12 weeks in the presence of control medium alone (con), non-activated macrophage-conditioned medium (NMCM), activated macrophage-conditioned medium (AMCM), activated macrophage-conditioned medium (AMCM) produced in the presence of 1 μM SC-236 (SC-AMCM) or activated macrophage-conditioned medium (AMCM), to which 1 μM SC-236 was added after production (AMCM ⁺ SC). The β-catenin doublet was 90-92 kDa in size. β-actin (42 kDa) was used as a loading control. The images of the blots are cropped but no bands have been omitted by editing. (H) TOPflash activity in control IEC-6 cells (con) or IEC-6 cells cultured in the presence of AMCM for 12 weeks. Data are TOPflash firefly luciferase values normalised to Renilla luciferase activity. No FOPflash luciferase activity was detectable (data not shown). Columns and bars represent the mean and SEM of triplicate values.