Extended Data Fig. 6: PGE2–Ptger4 drive the induction of Yap target genes in intestinal organoids.
From: Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche
a, Volcano plot displaying the results of differential gene-expression analysis performed in single epithelial cells from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures (n = 1,585). Yap1 and Yap target genes18 are indicated. Moderated t-test with false-discovery rate (Benjamini–Hochberg) correction. b, Expression levels of the genes indicated in single epithelial cells from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures (n = 1,585), projected onto t-SNE plots. c, Experimental setup for data shown in d, e. Crypts were grown into organoids or spheroids by 3D culture in OGM or OGM that was supplemented daily with 0.1 μM dmPGE2 for 7 days. Gene expression levels were measured by RT–qPCR on day 7. d, Relative expression of Yap target genes (Ly6a, Clu, Il1rn, Msln and Cxcl16) in day 7 organoids and PGE2-driven spheroids developed from wild-type crypts. N = 3 3D cultures per condition. Two-tailed Welch’s t-test. e, Relative expression of Yap target genes in day 7 organoids and PGE2-driven spheroids developed from crypts isolated from Ptger4f/f and Ptger4ΔIEC mice. n = 3 3D cultures per genotype and condition. One-way ANOVA. f, Correlation between the expression levels of metagenes of a Yap transcriptional program and an early (non-tumour) ApcMin/+ tumorigenesis transcriptional program in single epithelial cells (n = 1,585) from the Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures of Fig. 3. g, Small intestinal crypts were grown into organoids or spheroids with OGM or OGM that was supplemented daily with 0.1 μM dmPGE2. Western blot analysis for Yap1 and β-actin was performed in total lysates from untreated organoids, organoids treated with 0.1 μM dmPGE2 for 16 h and untreated spheroids. Data from one organoid and three independent spheroid cultures. h, Relative expression of the Yap1 gene and Yap target genes in wild-type organoid cultures treated with 0.1 μM dmPGE2 for 13 h, as determined by RT–qPCR. n = 3–5 cultures per condition. Statistical comparisons were performed with unpaired two-tailed t-test. For Ly6a, Welch’s correction was applied. i, Western blot analysis for Ser127 pYap and total Yap performed in total lysates from wild-type organoids stimulated with 0.1 μM dmPGE2 for the indicated time-points. Indicative of five independent experiments. j, Relative expression of Yap target genes in wild-type organoids treated with 1 μM verteporfin and 0.1 μM dmPGE2 for 13 h. n = 3–4 cultures per condition. Statistical comparisons were performed with unpaired two-tailed t-test, two-tailed Welch’s t-test or Mann–Whitney test on the basis of the criteria described in Methods. All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
