Fig. 3: Effect of loss of heterozygosity (LOH) on mutant cell behaviour.

a A 3D primary culture system was used to characterize p53 mutant cells in vitro. Oesophageal keratinocytes were isolated from transgenic mice and p53* mutation was induced by adenovirus carrying cre recombinase. b Cell competition assay. p53^*/wt and p53^*/− cells were co-cultured with p53^wt/wt cells respectively and relative fitness was examined. Representative immunofluorescence images from three biological replicates at day 0 and 28 are shown. Green, GFP; blue, DAPI. Scale bars, 50 µm. c Quantitation of cell competition assay by flow cytometry. Graph shows the fold change of proportion of GFP + p53^*/wt or p53^*/− cell in the culture. Black lines indicate mean and s.e.m. p value, two-tailed ratio paired t-test, n = 3 replicate cultures. d RNAseq analysis showing differentially expressed transcripts in p53 mutant cells which could affect the fitness of cells. RNAseq data were subjected to the Gene Ontology analysis (enrichGO in R package clusterProfiler). Enriched biological processes in pairwise of comparisons both p53^*/wt vs p53^wt/wt and p53^*/− vs p53^wt/− were considered to be affected by p53* expression. Heatmaps were generated for genes associated with the cell cycle, cell division (chromosome, microtubule, and spindle), small GTPase mediated signal transduction, and keratinocyte differentiation. n = 6 biological replicates per genotype. See Supplementary Data 8–11.