Unlicensed G₁ is lost in Apc mutant epithelium. (A) A representative vibratome section of Apc^Min/+ intestinal epithelium stained with Hoechst (nuclei), phalloidin, and an antibody against Mcm2. Bar, 200 µm. Regions of normal histology and a region containing a polyp are highlighted. (B) Representative image of an extracted Apc^Min/+ isolated crypt (i) stained with Hoechst (nuclei) and an antibody against Mcm2 (red) after a 1-h EdU pulse (green). A representative image of an abnormally elongated crypt is displayed (ii) showing a clonal ribbon of cells in the distal TA compartment, which are EdU⁺. Quantification of cell-cycle stages across the crypt axis (Fig. 3) is shown (iii; n = 40 crypts). Bars, 10 µm. (C) Representative bright-field images of organoids cultured from WT and Apc^Min/+ mice that have undergone LOH (Apc^Min/Min). Bars, 50 µm. (D) Representative images of unextracted and extracted Apc^Min/Min organoids stained with Hoechst (nuclei) and an antibody against Mcm2 (red) after a 1-h EdU pulse. Bars, 50 µm. (E) Representative flow cytometry profiles from cells of extracted WT and Apc^Min/Min organoids showing DNA-bound Mcm2 versus DNA content (i). The 2N G₁ cells of the profile shown are displayed (ii), showing unlicensed G₁, early G₁, and peak (fully licensed) G₁ populations. Data are representative of three independent experiments. (F) Quantification of the populations described in E(ii). Data are displayed as means ± SEM (n = 3 organoids. (G) Western blot of Rb and pRb (low and high exposure) levels between WT and Apc^Min/Min organoids. Two bands, corresponding to hypo- and hyperphosphorylated Rb, are shown. (H) Quantification of the fold increase of pRb:Rb for Apc^Min/Min organoids (n = 3) and polyps isolated from Apc^Min/+ mice (n = 6), compared with WT organoids and tissue.