Fig 2: Tipe0−/− mice have inappropriate basal activation of the injury-response stem cell program, but an inability to induce the program to regenerate.a Sca-1 staining in Tipe0−/− spheroids generated by depriving growth factor withdrawal; bars = 130 µm; insert 3× magnified. Representative of 3–4 Tipe0−/− spheroids/experiment, with three independent replicates. b Flow cytometry for Sca-1+/Epcam+ cells. N = 3, representative of 2 independent experiments. p = 0.0005 two-tailed t-test with Welch′s correction vs WT. c IF for Sca-1 and E-cadherin in healthy WT and Tipe0−/− tissue. E-cadherin was used as an enterocyte counterstain. Images representative of the mean intensity in 4 mice/group, with two fields of view/mouse randomly selected; bars = 130 µm; arrows mark enterocytes staining for Sca-1; insert 3× magnified. Sca-1 staining linearly enhanced in both images for easier merged channel visualization. d Quantification of enterocyte Sca-1 fluorescence intensity from c; N = 4 mice/group; p = 0.0286. e Sca-1 and E-cadherin staining from mice given DSS, with quantitation (f). E-cadherin is an used as an enterocyte counterstain. Quantification of total Sca-1 signal was by relative fluorescence intensity (RF), normalized to WT. N = 7 crypts/per mouse from 4 mice/group. Images representatives 3/4 mice evaluated for each group; bars = 70 µm; insert 3× magnified. g Colonic Sca-1 levels by RT-PCR. N = 7\(WT and Tipe0−/− healthy), N = 8 (WT DSS), N = 6 (WT and Tipe0−/−). Multiple group comparisons were by Kruskal–Wallis one-way ANOVA. Two-tailed Mann–Whitney U test was used to confirm ANOVA findings. h YAP staining in mice given DSS; bars = 70 µm, insert bars = 20 µm. Arrows show areas of high nuclear colocalization. Images representative of eight WT and five Tipe0−/− mice evaluated. i Quantification of nuclear YAP from h by Intensity Correlation Quotient (ICQ). N = 8 (WT), 5 (Tipe0−/−); p = 0.0025. j WB for pYAPS127 and YAP, with densitometry (N = 4/group); p = 0.0102 for YAP, p = 0.0021 for pYAPS127 by one-sample two-tailed t-test. For all graphs, *p < 0.05; **p < 0.01; p < 0.001; ****p < 0.0001 vs WT, or as indicated; error bars show mean ± SEM. Analyses done by two-tailed Mann–Whitney U test unless otherwise specified. Source data are provided as a source data file.

Fig 3: Co‐culture with cancer cells induces an activation of ECs via inflammatory signalling pathways. (a), Schematic of the in vitro coculture system used to study the effect of TME generated by human breast cancer cells (MDA‐MB‐231) on human endothelial cells (HUVECs). HUVECs were cultured with/without MDA‐MB‐231 for 48 h (i.e. coculture vs. monoculture), followed by endothelial cell isolation using magnetic beads coated with anti‐CD31 antibody. Then, RNA‐Seq was performed to assess transcriptomic changes induced by MDA‐MD‐231. (b), Flow cytometry analysis of HUVEC, MDA‐MB‐231 and cocultured HUVEC/MDA‐MB‐231 cells stained for the CD31 endothelial marker and EpCAM. (c), Western blotting of endothelial marker CD31 and breast cancer marker EpCAM in lysates from HUVECs purified from mono or cancer cell coculture and from MDA‐MB‐231 cells. HSP70 is a loading control. (d), Selected gene sets significantly enriched in co‐ versus mono‐HUVECs from GSEA of RNA‐Seq data obtained as in (a). (e), GSEA enrichment of inflammation‐associated pathways in co‐HUVECs. (f), Expression levels (log2TPM) of several adhesion molecules in HUVECs monocultures or in HUVECs cocultured with MDA‐MB‐231 as shown by RNA‐Seq data. TPM = Transcripts per Million. (g), RT‐qPCR analysis of expression of the adhesion molecules ICAM‐1, VCAM‐1 in HUVECs cocultured with MDA‐MB‐231 relative to HUVECs alone. Data are mean ± SEM. **P < 0.01 calculated by One‐Sample T‐test (n = 6)