Figure 7. Smad4 mediates the negative regulation of BMP on Lgr5⁺ stem cell self-renewal.

(a) Olfm4 in situ hybridization and Sox9 staining in intestines from 1-month-old Smad4^fl/fl and Vil-Cre;Smad4^fl/fl mice. Images are representative of n=5 mice per genotype. (b) Ki67 and lysozyme staining in intestines from 1-month-old Smad4^fl/fl and Vil-Cre;Smad4^fl/fl mice. Dotted lines delineate the region of crypts. Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Images are representative of n=5 mice per genotype. (c) Intestinal organoids derived from Smad4^fl/fl and Smad4^KO (Vil-Cre;Smad4^fl/fl) mice were cultured in ENR (EGF, Noggin and R-spondin) or ER medium for 5 days and photographed. Images are representative of three independent experiments. (d) Intestinal organoids derived from Smad4^fl/fl and Smad4^KO (Vil-Cre;Smad4^fl/fl) mice were treated with BMP4 for 36 h followed by Olfm4 in situ hybridization, Sox9 staining and Ki67 immunofluorescence staining. Nuclei were counter-stained with DAPI. Images are representative of three independent experiments. (e,f) Smad4^fl/fl and Smad4^KO (Vil-Cre;Smad4^fl/fl) organoids were treated with BMP4 for 4 h and the mRNA level of indicated genes were measured by quantitative RT–PCR. Data represent mean±s.e.m of three independent experiments. *P<0.05, ***P<0.001 by Student's t-tests. Scale bars, 50 μm.