Extended Data Fig. 6. The key pathways for RANK-induced stem cell exhaustion in vivo.

a-c, Alterations in the NF-κB, anti-apoptotic, and BMP pathways in jejunal organoids from caRANK^vil-Tg mice (n = 4) compared to organoids from control mice (n = 4). Upper panels show GSEA enrichment plots. Bottom panels show heatmaps of the top 15 genes upregulated in caRANK^vil-Tg organoids. Expression profiles of RANKL stimulated jejunal organoids were compared to non-stimulated (control) jejunal organoids cultured for the same time period. Total RNA was isolated from small intestinal organoids generated from two weeks old control and caRANK^vil-Tg littermate (n = 4 for each group) and processed for RNA-seq. d, Differential gene expression analysis of RNA-seq data from mouse jejunal organoids derived from caRANK^vil-Tg (n = 4) and control (n = 4) mice. Normalized CPM values of selected transcripts of anti-apoptotic genes, stem cell signature genes and BMP signalling genes are shown. e, Western blotting of phosphorylated IκB-α and total IκB-α in isolated intestinal epithelial cells from 3 wks old control (n = 3) and caRANK^vil-Tg (n = 3) mice. β-Actin is shown as a loading control. For gel resource data, see Supplementary Fig. 1. f, Schematic outline of the proposed role of RANK/RANKL-induced stem cell proliferation and exhaustion. The figure was created with BioRender.com. g, Ratios of the numbers of organoids derived from Traf6^WT and Traf6^ΔVil mice after prolonged culture in the presence of rmRANKL (50 ng/ml). Numbers of organoids were counted at each passage. The ratio of organoid numbers in the RANKL Traf6^WT group was normalized to control Traf6^WT organoids, whereas the ratio of organoid numbers in the RANKL treated Traf6^ΔVil group was normalized to untreated control Traf6^ΔVil organoids. Data were combined from two independent experiments. n = 11 (Traf6^WT), n = 11 (Traf6^WT + RANKL), n = 11 (Traf6^ΔVil), n = 11 (Traf6^ΔVil + RANKL). h, Average length of villi and average number of OLFM4⁺ cells in each crypt per mouse from the indicated mice in the indicated mice described in Fig. 2d,e. i, Kaplan–Meier survival curve of control (n = 14), caRANK^vil-Tg (n = 8), Apc^min/+ (n = 13), and caRANK^vil-Tg, Apc^min/+ (n = 11) mice. j, Numbers of macroscopic adenomas and k,l, tumour diameters in the small intestine and colon of 4 months old control Apc^min/+ (n = 5 mice) and caRANK^vil-Tg, Apc^min/+ (n = 5) mice. Small intestines were divided equally into 4 parts from the proximal (duodenum, labelled 1) to the distal (ileum, labelled 4) and adenomas assessed for each region. Total tumour numbers in Apc^min/+ mice; n = 29 in region 1, n = 53 in region 2, n = 156 in region 3, n = 231 in region 4, n = 469 in total, n = 14 in colon, and in caRANK^vil-Tg, Apc^min/+ mice (n = 29/24/29/37/119/23). Each data point in k represents the measurements of the tumour diameter in individual tumours and in l represents the average measurement of the tumour diameters per mouse. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Enrichment-adjusted p-values (p-value), False Discovery Rates (FDR) and Normalized Enrichment Scores (NES) were calculated using two sided-fGSEA (a-c). Two-sided DESeq2 Wald tests, adjusted with the Benjamini–Hochberg procedure (d). One-way analysis of variance (ANOVA) with Tukey’s post hoc test (g); Two-tailed Mann–Whitney U-test (h,j,l); Kaplan–Meier survival curve with a log-rank test (i); Two-tailed Student’s t-test (k).

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