Fig. 2: Epithelial cell-intrinsic activation of TGFβ signalling is tolerated in vivo, but not in vitro.
a Representative images of small intestinal organoids derived from uninduced VilCreER;Alk5CA (Alk5CA) and VilCreER;Alk5+/+ (Alk5+/+) mice, following 5 days of in vitro treatment with vehicle control (EtOH for 4-hydroxytamoxifen; PBS/BSA for TGFβ1), 4-hydroxytamoxifen (Tmx, top right panel) or TGFβ1 (bottom right panel). n = 3 organoid lines per group. Treatments were repeated twice. Scale bar, 50 μm. b Relative viability (dead; grey vs alive; blue) of Alk5CA and Alk5+/+ organoids measured 5 days after the indicated treatments. Alk5CA organoids were treated with EtOH, Tmx, or Tmx plus ALK5i, whereas Alk5+/+ organoids were treated with PBS/BSA or TGFβ1. Representative images of treated organoids, with or without ALK5i, are shown in Supplementary Fig. 2a. n = 3 biological replicates per group, each in technical triplicate. Data were ±s.e.m; *P = 0.05, one-tail Mann–Whitney U-test. c qPCR for TGFβ-target genes expressed by organoids described in a. n = 3 biological replicates per group; EtOH (green), tamoxifen (yellow), PBS/BSA (grey), TGFβ1 (red) each in technical triplicate. Data were ±s.e.m; *P = 0.05, one-tail Mann–Whitney U-test. d Representative IHC (p-SMAD3, cleaved caspase-3 (CC3), BrdU and lysozyme) and ISH (Olfm4) in the small intestine of VilCreER;Alk5+/+ (Alk5+/+) and VilCreER;Alk5CA (Alk5CA) mice 4 days post-tamoxifen induction. Arrows indicate CC3-positive apoptotic cells. Scale bar, 100 μm. e–h Quantification of small intestinal epithelial cells stained for cleaved caspase-3 (CC3) (e), BrdU (f), Olfm4 (g), and lysozyme (h) cells from mice of the indicated genotypes 4 days post-tamoxifen induction. n = 3 mice per group and *P = 0.05, except for BrdU scoring; n = 5 Alk5+/+;Smad4+/+, n = 6 Alk5CA;Smad4+/+ and n = 4 Alk5CA;Smad4fl/fl mice, *P = 0.03. All data were ±s.e.m; two-tail Mann–Whitney U-test.
