Extended Data Fig. 4: Generation and characterization of novel Notum conditional knockout.
From: NOTUM from Apc-mutant cells biases clonal competition to initiate cancer
a, Schematic of the Notum locus and recombined Notumc allele with relevant genome editing sites indicated. b, Southern blot analysis of embryonic stem (ES) cell Notumc clones and WT genomic DNA showing successful recombination at the Notum locus (4.7-kb product). The 13.6-band and 4.7-kb band represent endogenous and recombined alleles, respectively (arrows). c, Schematic of tamoxifen treatment regimen and tissue analysis 7 and 14 d.p.i. of Lgr5CreER;Apcfl/fl;Notum+/+ (NotumWT) and Lgr5CreER;Apcfl/fl;Notumc/c (NotumcKO) mice (top). Representative images of small intestinal sections stained for β-catenin from NotumWT and NotumcKO mice 14 days following induction with 120 mg/kg (3 mg) tamoxifen are also shown (bottom). The arrows indicate dysplastic crypts with nuclear β-catenin (magenta border). The boxed area shows a close-up of β-catenin+ crypts. Sections from n = 4 mice per genotype were stained. Scale bar, 50 μm. d, Representative agarose gel electrophoresis of products from conventional PCR showing relative recombination of Apcfl and Notumc alleles 5 days after tamoxifen induction. The 250-bp and 513-bp bands represent recombined Apc and Notum, respectively. For gel source data, see Supplementary Fig. 2. n = 3 mice. e, Quantification of small intestinal β-catenin+ lesions in NotumWT and NotumcKO mice, induced with 3 mg tamoxifen, and sampled at 7 and 14 d.p.i. n = 4 per genotype at 7 d.p.i; n = 18 NotumWT mice and n = 12 NotumcKO mice at 14 d.p.i. f, Quantification of large intestinal β-catenin+ lesions at 14 d.p.i (n = 18 NotumWT mice, n = 12 NotumcKO mice). g, Representative images of β-catenin IHC depicting fully and partially fixed Apc-mutant crypts in NotumWT and NotumcKO mice, respectively (left). Mice were induced with 3 mg tamoxifen and sampled at 14 d.p.i (n = 4 per genotype). The ratio of fully to partially fixed crypts in NotumWT and NotumcKO mice is also shown (right). Scale bar, 100 μm. h, Relative percentages of clonal crypt classification (clonal crypt phenotype) from mice described in g. i, Analysis of NotumWT and NotumcKO adenomas at 14 d.p.i. Representative confocal images (left). Ki67 (magenta), Lgr5–EGFP (green), nuclei (cyan), β-catenin (white) and adenomas (yellow dashed line). Quantification of Lgr5+ cell frequency within β-catenin+ Apc-mutant clones (middle). Proliferation of Lgr5+ and Lgr5− Apc-mutant cells (β-catenin+) (right). n = 5 mice/genotype. Scale bar, 20 μm. In the box plots, the line represents the median, the box shows the interquartile range and the whiskers represent the range. Data are mean ± s.e.m. In e–g, i, Mann–Whitney two-tailed U-test; P values are shown in the corresponding panels.
