Fig. 5: p53^*/wt mutant clones in mutagenized epithelium.

a Protocol: p53^*/wt mice were treated with DEN or vehicle followed by clonal induction, oesophagus samples were collected at indicated time points (triangles). b Projected confocal z-stacks showing p53^*/wt clones in epithelial wholemounts. Representative images from two independent experiments. 4 weeks, n = 4 control and n = 3 DEN mice; 12 weeks, n = 2 mice per group; 52 weeks, n = 6 control and n = 4 DEN mice. Green, GFP; Red, KRT6; blue, DAPI. Scale bars, 500 µm. c Average proportion of projected labelled area. Number of mice as in b. Error bars, mean ± s.e.m., p values by two-tailed unpaired t-test with Welch’s t correction. See Supplementary Data 16. d Schematics of p53^*/wt clone behaviour in DEN-treated tissue. p53^*/wt outcompetes wild type cells, but not all DEN-induced mutant cells. Many p53^*/wt clones are lost in 4-weeks. Surviving p53^*/wt clones vary widely in size and are found in both epithelium and tumours. p53^*/wt clone can be outcompeted by surrounding fitter clones (black arrows) or itself be fitter than adjacent mutant clones (white arrows). e Protocol: Mice were treated with DEN or vehicle after colonization by p53^*/wt mutant cells. Triangles, sampling time points. f Top down views of basal layer of wholemounts from p53^*/wt induced mice 4 weeks after DEN treatment (n = 9 mice) compared to control (vehicle, n = 4 mice). Green, GFP; red, KRT6; blue, DAPI. Arrowheads indicate large nuclei in p53^*/wt clone area following DEN treatment. Scale bar, 50 µm. g Sequencing analysis at 1 year time point (protocol: e). DNA extracted from 2 mm² grid biopsies were subjected to ultradeep targeted sequencing. Tumours (orange circles) were microdissected out from the epithelium prior to the following sequencing analysis. n = 40 (control) and 42 (DEN) biopsies from 2 mice per condition (h−l). h Number of mutations. Every dot corresponds to a sample. i Estimated mutation burden. p value by two-tailed unpaired Student’s t-test. Error bars, mean ± s.e.m. j, Mutational spectrum of DEN-treated p53^*/wt epithelium. k Percentage of mutation types identified in each condition. l Positively selected somatic mutations in DEN-treated samples. dN/dS ratios for missense, truncating (nonsense + splice) and indels. See Supplementary Data 14 and 15.