Figure 3: Impaired DNA damage-induced p53 activation in CYLDΔ932 mutant epithelial cells.

(a) Representative pictures from TUNEL-stained colon sections from the indicated mice 8 h after AOM or PBS injection and quantification of TUNEL-positive IECs in colonic crypts. Data shown as mean±s.e.m. of 150 well-oriented crypts from three animals of each genotype. Statistical significance was determined with Student’s t-test; *P≤0.05. (b) Immunoblot analysis for caspase-3 (C3) and cleaved caspase-3 (CC3) in CpT-treated intestinal organoids prepared from the indicated mice. (c) FACS quantification of CpT-induced cleaved caspase-3-positive cells in organoids from the indicated mice. Mean±s.d. of biological triplicates shown. Statistical significance was determined with Student’s t-test **P≤0.005. One representative out of three independent experiments is shown. (d) Immunohistochemical analysis for active caspase-3 in CpT-treated intestinal organoids from the indicated mice. Arrows indicate epithelial cells stained for active caspase 3. Note that the luminal contents of organoids show unspecific staining. Scale bar, 50 μm. (e–h) Immunoblot analysis of p53, p21 and tubulin and qRT-PCR analysis of p53 target gene expression in CpT-treated intestinal epithelial organoids (e,f) and primary epidermal keratinocytes (g,h) prepared from the indicated mouse lines. Graphs in f and h show means ±s.d. of the absolute values of technical duplicates, from one representative out of two independent experiments. qRT-PCR, quantitative real-time PCR.