Fig. 1: NOTCH1 mutant clones in human esophageal epithelium.

a, Cyrosection of human esophagus. NOTCH1 (green) stains basal and lower suprabasal layer cells, expression is lost in regions of the esophagus. F-actin, magenta; Pa, papillae. Dotted line indicates epithelial submucosal boundary. Image representative of three donors. Scale bar, 100 µm. b, Protocol for c–f. Cryosections were stained for NOTCH1. Contiguous NOTCH1⁺ and NOTCH1⁻ staining areas were microdissected and sequenced. c, Representative images from b for donor PD40290. NOTCH1 is red, DNA is blue. Upper labels show sample identification (Id) and NOTCH1 staining status (positive, + or negative, −) for each sample. Lower labels show nonsynonymous NOTCH1 mutations and VAF and indicate CNLOH if detected. Only mutations with VAF > 0.1 are displayed. Mutation effects are color coded (indel_splicing, gray; missense, blue; nonsense, red). Dashed lines delineate the epithelium and submucosa (white) and borders of sequenced samples (yellow). Solid lines separate the two images of the adjacent regions. Scale bars, 250 µm. d, Results from b, showing NOTCH1 staining, donor identification, NOTCH1 mutation calling, CNLOH affecting NOTCH1 locus and number of NOTCH1 mutations per sample (n = 86 samples from six donors aged 43–78 years). e, Proportion of missense, nonsense, indel/splicing or intronic/silent NOTCH1 mutations in NOTCH1⁺ and NOTCH1⁻ samples. Number of NOTCH1 mutations for each group is shown in brackets. f, Proportion of NOTCH1 mutant samples carrying monoallelic or biallelic NOTCH1 alterations in each donor. ‘Biallelic with second mutation’ category includes samples without CNLOH, carrying at least two mutations with VAF ≥ 0.15. Numbers in brackets are total number of NOTCH1 mutated samples per donor. g, NOTCH1 (green, upper panel) and NICD1 (red, lower panel) staining in successive sections of epithelium from an aged donor. ITGA6 (magenta) marks the basal cells. DNA is blue. Inset shows basal and lower suprabasal cells (white rectangles). Dashed lines delineate staining pattern. Images representative of six middle-aged and elderly donors. Scale bar, 100 µm. h, Proportion of tissue positive or negative for NOTCH1 and NICD1 in donors aged 20–78 years (total section length 4774–17988 µm per donor, n = 9 donors). Id, identification. See Supplementary Tables 1–5.