Fig. 1. APC^Min/RasV12-transformed cells are basally delaminated in a non-cell autonomous fashion.

a, c, e, f Confocal microscopic images of intestinal villi (a) or intestinal organoids (c, e, f) from APC^Min-Villin-GFP, Villin-RasV12, or APC^Min-Villin-RasV12 mice. In a, APC^Min-Villin-GFP, Villin-RasV12, or APC^Min-Villin-RasV12 mice were injected with low-dose tamoxifen and were sacrificed 3 days later. Frozen sections were stained with DAPI (blue), anti-GFP antibody (green), and phalloidin (red). In c, e, f, intestinal organoids from APC^Min-Villin-GFP, Villin-RasV12, or APC^Min-Villin-RasV12 mice were treated with 100 nM (c, e) or 1 μM tamoxifen (f) and cultured for the indicated times (d) or 24 h (e, f). Ba and Ap stand for the basal and apical sides, respectively (a, c, f). White squares in the left images are magnified in the corresponding right panels (a, c, f). White arrowheads depict the apically extruded cells whereas white arrows indicate the basally extruded cells, respectively (a, c, e). In (f), An arrow indicates a single transformed cell that is basally extruded (left magnified image) while the clustered transformed cells that are not extruded are depicted by an arrowhead (right magnified image). Scale bars, 50 μm (a, c), 100 μm (e), and 10 μm (f). b, d, g Quantification of apical and basal extrusion of transformed cells in small intestine (b) or intestinal organoids (d, g). ‘Not extruded’ indicates cells remaining within epithelium. ‘Apical extruding’ indicates apically extruding cells, with their nucleus apically shifted, that are still attached to the basement membrane. ‘Apical extruded’ indicates cells completely detached from the basement membrane and ‘Basal extrusion’ indicates cells delaminating into the basal lamina. Data are mean ± s.e.m. n = three independent experiments. *P = 0.0054 for basally extruded cells between RasV12- and APC^Min/RasV12-expressing cells (b), unpaired two-tailed t-test. In g, the clusters are categorized by the number of APC^Min/RasV12 cells present in each cluster. Data are mean ± s.e.m. n = three independent experiments. *P = 0.0037; **P = 0.0007 for cells with extrusion, unpaired two-tailed t-test.