Figure 2. Effect of ICAD deficiency on colony formation in soft agar by fibroblasts exposed to IR and on TNF+CHX+BA or DMH-induced death in CECs.

(A) ICAD^−/− and WT mouse fibroblasts were either exposed to IR (2 Gy) or sham-irradiated, after which they were cultured in soft agar with complete medium for 3 weeks. Colonies were then counted with an automated counter. Data are expressed as a percentage of the value for sham-irradiated WT cells and are means±SD of triplicates from a representative experiment. *, difference from sham-irradiated WT cells, p<0.05; #, difference from WT cells exposed to IR (2 Gy), p<0.05. (B) CECs were prepared from WT mice and allowed to propagate for 4 days. Cells were then fixed and subjected to immunofluorescence with antibodies to pan-cytokeratin (red) or staining with DAPI (blue); top panel is an example of CECs visualized by bright field microscopy. Yellow arrows indicate colonic crypts. (C) CECs, isolated from WT or ICAD^−/− mice, were treated with 2.5 ng/ml TNF combined with 5 µg/ml cyclohexamide and 3 mM butyric acid (designated as TNF+CHX+BA) or 1 mM DMH. Cells were incubated for 48 h in the absence or presence of TNF+CHX+BA or DMH, after which, after which cell viability was assessed by measurement of calcein-AM fluorescence. Data are expressed as a percentage of the viability of untreated cells and are means±S.D. of values from four wells from a representative experiment. *, Difference from respective control, p<0.05. #, Difference from respective treated cells, p<0.05. (D) Mice received an ip injection of 20 mg/kg DMH or saline (control). Mice were sacrificed after 48 h, and their distal colons were removed and formalin-fixed. Tissue sections were then prepared and subjected to H&E staining or TUNEL staining. Pycnotic/apoptotic cells were determined by nuclear condensation, cellular fragmentation, shrinkage, and/or detachment from mucosa, aided with TUNEL assay. On average, 40 crypts per section were counted. Results are expressed as the number of pycnotic/apoptotic cells per mm². Data are means±SD of values from at least six mice per group. *, difference from the respective untreated mice; p<0.01; #, difference from WT mice exposed to DMH, p<0.01. (E) Mice were treated as in (D) except that they were sacrificed after 24 h. Distal colons were removed and formalin-fixed. Tissue sections were then prepared and subjected to IHC with antibodies to poly(ADP-ribose). Red boxes indicate the magnified regions; yellow arrows show poly(ADP-ribose)-immunoreactive nuclei. (F) A quantification of poly(ADP-ribose)-immunoreactive nuclei of CECs in the colonic mucosa of the different experimental groups.