Fig. 4

Activated STAT6 promotes proliferation of IECs in VillinCre_STAT6vt mice but does not affect the number of stem cells. a Naive VillinCre_STAT6vt and their control litter mates were intraperitoneally injected with BrdU. After 24 h, ileal pieces were collected and cryosections were stained for BrdU (red) and nuclei were counterstained with DAPI (blue). Proliferation was determined by measuring the distance from crypts to the most distant BrdU⁺ cell on the villus axis. In addition, the percentage of proliferating cells was analyzed by counting all BrdU⁺ cells per villus. b Length of the whole small intestine of naive VillinCre_STAT6vt mice (dark gray bar) and their control littermates (light gray bar). c mRNA expression of Lgr5, Dll4, Pofut1, Hes1, Spdef, Math1, Sox-9, and Sox-4in the ileum of naive VillinCre_STAT6vt mice (black bar) and their control littermates (gray bar) was analyzed by quantitative RT-PCR. HPRT1 was used as the reference gene for normalization. d Small intestinal organoids were generated for up to 7 days using crypts from naive VillinCre_STAT6vt mice (top) and their control littermates (bottom). Growth and differentiation was documented every day by light microscopy. e Organoids from 12 wells per mouse were pooled and analyzed by western blot for total STAT6 (upper row) and phosphorylated STAT6 (pSTAT6, middle row). β-Actin served as reference protein. Numbers indicate the molecular weight in kD. f Organoids from 4 wells per mouse were pooled on day 7 of culture, RNA was isolated, and mRNA expression of Villin, Retnlb, DCLK1, IL-25, and Ang4 genes was analyzed by quantitative RT-PCR. HPRT1 served as the reference gene for normalization. Pictures in a, d are representative of one out of two independent experiments. Representative pictures in d show organoids on days 2 and 7 of culture. Bar graphs show the mean + s.e.m of pooled data from two independent experiments a–c or show the mean + s.d. of one experiment f. The western blot is representative for two mice per group from one experiment e. Two to three mice per group per experiment were used. Scale bars represent 20 µm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, unpaired two-tailed Student’s t test