Fig 2: Keratinocyte cell cycle exit and initiation of differentiation is marked by a switch from APOBEC3B to APOBEC3A expression.(A) qRT-PCR-based gene expression measurements (n = 3) for APOBEC3A (P = 0.0002), KRT10 (P = 0.0002), IVL (P < 0.0001), APOBEC3B (P = 0.0001), MKI67 (P < 0.0001) and MCM7 (P = 0.0033) in proliferating NIKS (Async) or following 48 h of growth factor deprivation (Starved). (B) Representative cell cycle profiles of Async and starved NIKS measured by PI staining and flow cytometry. (C) qRT-PCR-based gene expression measurements (n = 3) for APOBEC3A (P = 0.0036), KRT10 (P = 0.0004), IVL (P = 0.0007), APOBEC3B (P = 0.9445), MKI67 (P = 0.0032) and MCM7 (P = 0.0001) following 24 h of vehicle control (DMSO) or 100 nM afatinib treatment (EGFRi). (D) Representative cell cycle profiles of DMSO and afatinib-treated NIKS measured by PI staining and flow cytometry. (E) qRT-PCR-based gene expression measurements (n = 3) for APOBEC3A (adjusted P values versus day 3 measurement: day 5 = 0.9960; day 7 = 0.0009; day 9 <0.0001), KRT10 (adjusted P values versus day 3 measurement: day 5 = 0.9518; day 7 = 0.0005; day 9 <0.0001), IVL (adjusted P values versus day 3 measurement: day 5 = 0.9983; day 7 = 0.0035; day 9 <0.0001), APOBEC3B (adjusted P values versus day 3 measurement: day 5 <0.0001; day 7 <0.0001; day 9 <0.0001), MKI67 (adjusted P values versus day 3 measurement: day 5 = 0.0001; day 7 <0.0001; day 9 <0.0001) and MCM7 (adjusted P values versus day 3 measurement: day 5 <0.0001; day 7 <0.0001; day 9 <0.0001) in NIKS collected 3, 5, 7, or 9 days after plating. (F) Representative cell cycle profiles of NIKS collected 3, 5, 7, or 9 days after plating were measured by PI staining and flow cytometry. (G) qRT-PCR measurements of APOBEC3A expression in primary human epidermal keratinocytes (NHEK) growing in FC medium and treated for 24 h with vehicle control (DMSO) or 100 nM afatinib (EGFRi, P < 0.0001) or following growth factor deprivation for 48 h (Starved, P = 0.0003). (H) Percentage of DDOST transcripts that were C > U edited at c558 in asynchronous growing NIKS (Async) and following 48 h of growth factor withdrawal (starved) measured by digital PCR assay (n = 3; P < 0.0001). Error bars = SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Pairwise comparisons were performed using unpaired two-tailed t-tests in (A, C, H). Comparisons of mRNA levels on days 5, 7 and 9 to day 3 in (E), and of mRNA levels in starved and afatinib-treated cells to DMSO-treated cells in (G) were performed using one-way ANOVA with Dunnett’s multiple comparisons test. Source data are available online for this figure.

Fig 3: APOBEC3B is predominantly co-expressed with proliferation markers MKI67 and MCM7 in cycling basal and suprabasal cells of the healthy human airway.Stacked barplot showing the proportion of each cell type in the single cell atlas of healthy human airways from Deprez et al that express each selected gene.

Fig 4: APOBEC3A and APOBEC3B are expressed in different subsets of tonsillar epithelial cells.(A) UMAP projection of 22,595 epithelial cells from oropharyngeal squamous cell carcinoma samples (n = 10; 18,619 malignant cells (red), 695 non-malignant cells (purple)), and matched normal tonsil (n = 7; 3281 cells (blue)). (B) GOBP terms enriched amongst the sets of 100 genes that were co-expressed with either APOBEC3A or APOBEC3B in 2649 (after QC) epithelial cells from normal tonsil. Terms ranked by P value calculated by EnrichR package (Fisher Exact test bias corrected using the z-score of the deviation from the expected rank). (C) UMAP projection depicting four phenotypes (basal, proliferating, differentiating, terminally differentiated) displayed by the normal tonsillar epithelial cells in our dataset. (D) Marker genes are used to identify the four epithelial phenotypes represented in (C). (E) Violin plots of gene expression in individual tonsillar epithelial cells, and UMAP projections of the density of gene expression in the tonsillar epithelial subtypes. (****P < 0.0001, Wilcoxon’s rank-sum test; Basal, n = 1047; Proliferating, n = 353; Differentiating, n = 1091; Terminally differentiated, n = 158).

Fig 5: APOBEC3A and APOBEC3B expression in scRNA-seq datasets representing normal and tumour epithelial cells from tissues in which cancers that display prominent APOBEC mutational signatures arise.The number above each pie chart represents the total number of epithelial cells in each dataset. The references for each dataset are provided in Table EV2.