Fig. 6. Tumors retain functional Notch1 in carcinogenesis.

a, Uninduced YFPCreNotch1^flox/flox mice were treated with DEN and SOR. Tissue was collected 28 weeks after treatment. Tumors were dissected from underlying submucosa and normal epithelium was cut into a gridded array of 2 mm² samples before targeted sequencing. Scale bar, 1 mm. b, Number of Notch1 mutations per amino acid is plotted by NOTCH1 protein domains in normal gridded biopsies (upper) and tumors (lower) from Notch1 wild type mice (normal, n = 115 biopsies from six mice; tumors, n = 17 biopsies from seven mice). Domains: EGF-like repeats, LNR, HD, TM, transmembrane, RAM, RBP-J-associated module, ANK, ankyrin repeats, TAD, trans-activation domain, PEST, rich in proline, glutamate, serine and threonine. c, dN/dS ratio for Notch1 mutations (top plot) and proportion of Notch1 mutant tissue in normal epithelium (purple bars) (n = 115 biopsies from six mice) and tumors (n = 17 biopsies from seven mice). Two-tailed P value, likelihood ratio test of dN/dS ratios². d, Representative NOTCH1 (magenta) and KRT14 staining (green) in tumors and surrounding tissue, DNA is blue. Image typical of 10 tumors from six animals. White dashed lines delineate tumor from adjacent normal tissue. Scale bars, 250 µm. e, Proportion of NOTCH1⁺ staining area in normal epithelium and tumors from the same control animals (each dot represents a mouse, n = 40 tumors from four mice). Two-tailed paired Student’s t test. f, Representative images showing nuclear NICD1 (magenta) in keratinocytes (KRT14, green) inside a tumor in comparison to the normal adjacent tissue. DNA is blue. Image typical of 10 tumors from six animals. Scale bars, 25 µm. g, Proportion of KRT14⁺ keratinocytes with nuclear NICD1 staining in tumors and surrounding epithelium in the same sections (each dot represents a tumor, n = 10 tumors from six mice). Two-tailed paired Student’s t test. See Supplementary Tables 19–23.