Figure 7: Paneth cell number increases in organoids overexpressing Sox9.

(a) Immunofluorescent stain for Sox9 (red), DAPI (blue) and EpCAM (green) is shown in organoids transduced with lentivirus expressing doxycycline-dependent inducible Sox9 (Sox9-Fuw). Sox9 (Sox9-Fuw Dox(+)) was induced by doxycycline (1 μg ml⁻¹) for 5 days; controls were cultured for 5 days without doxycycline. (b) Representative western blot for Sox9 in control and induced organoids is shown. Densitometry±s.d. was performed on three repeat western blots. **P<0.01; two-tailed t-test. (c) Sox9 mRNA (mean±s.d.) was assessed by qPCR from two experiments performed in triplicate in control and induced organoids. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. (d) Typical organoids from uninduced controls (left) and induced (right) cultures are shown. (e) PSA±s.d. of organoids (n=52 per group) are shown. **P<0.01; two-tailed Student’s t-test. (f) PSA±s.d. of buds (n=52 per group) are shown. **P<0.01; two-tailed Student’s t-test. (g) Paneth cells identified as granular cells using phase contrast microscopy within individual buds are shown. (h) Granular cell frequency (mean±s.d.) was estimated by dividing the total granular cell number by the total bud PSA (n=52 per group). **P<0.01; Mann–Whitney test. (i) Paneth cells were identified based on lysozyme stain (red) in cells delineated by EpCAM (green) for cell membrane and (DAPI) for nuclei. (j) Paneth cell frequency (mean±s.d.) was ascertained by dividing the number of lysozyme staining cells (red) delineated by EpCAM (green) by the total cell number (DAPI) per bud (n=35 per group). **P<0.01; Mann–Whitney test. (k) Relative expression of Defc-rs (mean±s.d.) was compared in uninduced control and Sox9-induced organoids by qPCR from two experiments performed in triplicate. Gapdh was used to control for loading. **P<0.01; two-tailed Student’s t-test. Scale bars, 50 μm.