Fig. 7: IL-18-Akt-Tcf4 signalling upregulates Lgr5⁺ stem cells.

a–c Flow cytometry analysis of CD24^-/low ileum crypts, isolated from the indicated uninfected or AIEC-infected mice at d6, for Lgr5⁺ stem cells. An isotype antibody was included to indicate the specificity of the Lgr5 antibody. d Immunofluorescence analysis of ileum crypts for Olfm4⁺ stem cells in the indicated uninfected or AIEC-infected mice at d6. Crypt base columnar (CBC, outlined with a solid line) stem cell and the above transit-amplifying (TA, outlined with a dashed line) compartments are indicated. e Flow cytometry analysis of IL-18-stimulated ileum organoids, derived from the indicated mice, for Lgr5⁺ stem cells. f Chromatin immunoprecipitation (ChIP) and PCR analyses of IL-22 or IL-18-stimulated wild-type ileum organoids for the binding of Tcf4 to the mouse Lgr5 promoter regions (P1~P6). Fold induction is calculated based on PCR signal strength of Tcf4-bound Lgr5 promoter P1 region before and after IL-22 or IL-18 stimulation. g Western blot and flow cytometry analyses of IL-18/Akt inhibitor-stimulated CMT93 cells for Akt phosphorylation and Lgr5. Quantification and the ratio (pAkt to total Akt, Lgr5 to β-actin) of protein bands are indicated. h ChIP and PCR analyses of IL-18/Akt inhibitor-stimulated CMT93 cells for the binding of Tcf4 to the mouse Lgr5 promoter regions (P1 and P5). Fold induction is calculated based on PCR signal strength of Tcf4-bound Lgr5 promoter P1 region before and after stimulation. Each symbol in bar graphs represents a well of CMT93 cell culture (g), an ileum crypt sample (a–c) or organoid culture (e, f) derived from one mouse. Data shown are representative (d, f–h) or combined (a–c, e) results from two independent reproducible experiments. Statistical significance is indicated using unpaired two-tailed t test (a–c), One-way ANOVA with Sidak’s multiple comparisons test (f, g), or Two-way ANOVA with Tukey’s multiple comparisons test (e). Data are presented as mean ± SD. Source data are provided as a Source Data file.