Fig. 4: E2Fs induce WNT ligands while RB family proteins reduce WNT ligands in CSCs.

a Schematic depiction of CRISPRi knockdown and CRISPRa induction of RB proteins in CSCs. b Overexpression of E2F1 and E2F4 induces WNT ligands whereas CDK4/6 inhibition reduces WNT ligands in CSCs. The heatmaps in (b, c) show the normalised mean mRNA expression of three biological replicates analysed by qPCR. c Overexpression of RB family proteins reduces but knockdown induces the expression of WNT ligands in CSCs. d E2F1/4 induce whereas RB family proteins reduce WNT4 transcription as revealed by WNT4 promoter-luciferase assays in CSCs. Data are presented as mean values ± SD. N = 3 independent experiments. Statistical analysis was performed by 1-way ANOVA with Dunnett’s multiple comparisons test. e RB family proteins lead to the reduction of β-catenin dependent transcription in CSCs as measured by the M50 Super 8x TOPFlash construct. Data are presented as mean values ± SD. N = 3 independent experiments. Statistical analysis was performed by 1-way ANOVA with Dunnett’s multiple comparisons test. f Schematic depiction of using primary tumour sample from PDAC patients. g WNT ligands have elevated expression in CSCs over non-CSCs that depends on CDK4/6 activity in primary tumour sample from PDAC patients. h Knockdown of RBs leads to the upregulation of EMT-inducing transcription factors. i Overexpression of RBs leads to the downregulation of EMT-inducing transcription factors. j, k Conditioned media from OE RB cells have slower wound healing whereas WNT ligand promotes wound healing as shown by ( j) brightfield images and k measurement of the unclosed wound area. Scale bar 50 µm. l Conditioned media from OE RB cells supports lower cell invasiveness compared to control OE GFP cell-conditioned media whereas WNT ligand promotes cell invasiveness. m WNT ligand expression is positively correlated with the expression of EMT-inducing transcription factors in PDACs. n Cell type annotation in PDAC patient tumour sample RNA-sequencing data. Epithelial cells mark cancer cells. o, p EMT-enriched and basal-like gene signatures in the PDAC patient tumours. Data are presented as mean values ± SD. Bar graphs depicted in (g, h, i, k, l) are n = 3 independent experiments. Statistical analysis in (g, h, i, k, l) was performed by 2-way ANOVA with multiple comparisons with Tukey correction. ns = not significant. Source data are provided as a Source Data file.