Fig. 1

WNT4 suppresses intestinal organoid branching. A RT-qPCR analysis of the interference efficiency of Wnt4 by siRNA in mouse small intestinal organoids. B Representative images (left) and budding numbers (right) of mouse small intestinal organoids transfected with either control siRNA (si-control) or Wnt4 siRNA (si-Wnt4) after 60 h. Scale bars, 200 μm. C RT-qPCR analysis of the overexpression efficiency of Wnt4 in mouse small intestinal organoids. D Representative images (left) and budding numbers (right) of mouse small intestinal organoids transfected with either vector control (vector) or Wnt4-overexpressing construct (Wnt4-OE) after 60 h. Scale bars, 200 μm. E Representative images of IF staining for Ki67 in mouse small intestinal organoids transfected with either control siRNA or Wnt4 siRNA. Scale bars, 50 μm. The percentages of Ki67-positive cells in the bud and non-bud areas of each organoid (n = 17 for si-control and n = 21 for si-Wnt4) were quantified separately. F Representative images of IF staining for Ki67 in mouse small intestinal organoids transfected with either vector control or Wnt4-overexpressing construct. Scale bars, 50 μm. The percentages of Ki67-positive cells in the bud and non-bud areas of each organoid (n = 16 for both Vector and Wnt4-OE) were quantified separately. G RT-qPCR analysis of the mRNA levels of stem cell marker genes (Lgr5, Ascl2 and Olfm4) in mouse small intestinal organoids after knockdown or overexpression of Wnt4. H RT-qPCR analysis of the mRNA levels of cell differentiation markers (Lyz, Muc2, Chga, Fabp1 and Fabp2) in mouse small intestinal organoids after knockdown or overexpression of Wnt4. All values are means ± SD. Statistical analyses were performed by unpaired Student’s t-test (A–H). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001