Figure 2A Quantification of the average expression ± s.e.m. of Ptger1, Ptger2, Ptger3, Ptger4, and Ptgir mRNAs in whole-thickness lung, ileum or colon tissues or in jejunal spheroids cultured in stem cell (Stem), WAE (dmPGE2) or enterocyte (EP4i) medium (n = 3 independent experiments). Data are presented as fold change compared to lung tissue. n.d., not detected.B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (B) Representative bright field images. Scale bars, 200 µm.B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (C) Quantification of average spheroid area ± s.e.m. relative to spheroids treated with DMSO alone (n = 4 images from two independent experiments).B-D Jejunal spheroids were cultured in differentiation medium with DMSO only or with 1 µM dmPGE2 and pharmacological inhibitors of EP1 (EP1i, SC 51322), EP2 (EP2i, PF 04418948), EP3 (EP3i, L-798,106) or EP4 (EP4i, L-161,982) at a concentration of 10 µM or an equivalent volume of DMSO vehicle. **p<0.01, ***p<0.001, ****p<0.0001 compared to the DMSO only group by one-way ANOVA and Dunnett's post-test. (D) Quantification of the average expression ± s.e.m. of Cldn4 mRNA relative to stem cellspheroids (n = 3 independent experiments).E, F Tat-Cre mediated recombination of Ptger4flox/flox (fl/fl) jejunal spheroids to generate Ptger4flox/Δ (fl/Δ) and Ptger4Δ/Δ (Δ/Δ) spheroid lines. (E) Schematic and representative PCR genotyping results. M, marker lane. bp, base pairs. (F) Quantification of the average expression ± s.e.m. of Ptger4 mRNA in spheroids cultured in stem cell media relative to fl/fl genotype (n = 3 independent experiments). **p<0.01, ****p<0.0001 by 2-way ANOVA and Dunnett's multiple comparisons post-test with the EP4i-treated fl/fl group set as the control.G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (G) Representative bright-field images. Scale bars, 200 µm. (H) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from two independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.G-I Ptger4flox/flox, Ptger4flox/Δ and Ptger4Δ/Δ spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (I) average expression ± s.e.m. of Cldn4 mRNA (n = 3 independent experiments) relative to EP4i-treated Ptger4flox/flox spheroids.J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (J) Representative bright-field images. Scale bars, 200 µm. (K) Quantification of average spheroid area ± s.e.m. (n = 4 images per group from 3 independent donor lines examined over 3 passages each) relative to stem spheroids.J-L Humanileal spheroids were cultured in differentiation medium with 1 µM dmPGE2 or 10 µM EP4i. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 1-way ANOVA and Tukey's post-test. (L) the average expression ± s.e.m. of CLDN4 mRNA (n = 3 independent donor lines) relative to stem spheroids.