Fig. 3: E2F1/4 and RB family proteins bind to regulatory regions near WNT ligand loci in CSCs.

a Principal Component Analysis of ATAC-seq data in A13A CSCs and non-CSCs. b Hierarchical clustering analysis of correlation coefficients for gene expression in RNA-seq samples. c ATAC-seq peak number in A13A non-CSCs and CSCs. d The distribution of ATAC-seq peaks in the genome. e The overlap of genes with peaks near their loci in A13A non-CSCs and CSCs. f E2F1 and E2F4 binding motifs are present around WNT loci based on ATAC-seq data analysis in pancreatic CSCs. g E2F4 and E2F1 consensus binding motifs at total ATAC-seq peaks. h Expression of RBs, E2F1/4, CDKs, Cyclin D1 and Cyclin D3 in the cell types in PDAC patient samples analysed by single-cell RNA-seq. and FZD5/6 expression in PDAC patient tumours based on single-cell RNA-sequencing. i E2F1 and E2F4 binding to WNT loci based on ChIP-seq data. j Representative images of H6C7 wt and H6C7 KRAS (G12V) cells. Live cells were visualised by fluorescence microscopy for detecting eGFP signal in addition to brightfield images. Scale bar 50 µm. k Schematic depiction of the H6C7 non-cancerous pancreatic ductal cells and the introduction of a KRAS (G12V) oncogenic mutation which is an early hallmark mutation of pancreatic ductal adenocarcinoma. The KRAS (G12V) cells also express eGFP. l CDK4/6-mediated phoshorylation of pRb protein is increased in cells with constitutively activated KRAS. m KRAS (G12V) increases WNT ligand expression in H6C7 cells. n E2F4 and E2F5 bind to the regulatory regions near WNT ligand loci in H6C7 wt cells. o pRb and RBL2 binding to WNT ligand loci is attenuated by KRAS mutation. p Schematic depiction of the CSC model system used in the following experiments. q Immunofluorescence imaging of cancer stem cell markers OCT4, CD133 and SSEA4 in CSCs. r E2F4 and E2F1 bind to the regulatory regions near WNT ligand loci in pancreatic CSCs. s RB family proteins bind to WNT ligand loci in CSCs. Data are presented as mean values ± SD. All bar graphs depicted in this figure are n = 3 independent experiments. Statistical analysis was performed by 2-way ANOVA with multiple comparisons with Tukey correction. Source data are provided as a Source Data file.