Promotion by LPA of the development of intestinal organoids.

(A) Isolated crypts were cultured with or without (control) various growth factors or lipid mediators indicated for 4 days. Scale bar, 50 μm. (B) Areas of intestinal organoids cultured as for (A) were determined. (C) The number of buds per organoid cultured as for (A) was determined. Quantitative data in (B) and (C) are means ± SEM for a total of 90 organoids per group in three separate experiments. **, P PD) Intestinal organoids cultured with or without (control) LPA or EGF for 4 days were subjected to immunostaining with antibodies to Ki67 (red) and to E-cadherin (green), and to staining of nuclei with DAPI (blue). The boxed areas in the upper panels are shown at higher magnification in the lower panels. Scale bars, 50 μm. (E) The number of Ki67-positive (Ki67+) cells normalized to the circumference of each organoid was determined. Quantitative data in (E) are means ± SEM for a total of 30 organoids per group in three separate experiments. *, P P < 0.01 (ANOVA and Tukey’s test).