Fig. 4

Exogenous NGF amplifies Wnt signaling in intestinal stem cells. a Immunofluorescent staining for β-catenin (red) in the sections of proximal colons from control and NMS mice treated with or without intraperitoneal injection of NGF and MNAC13 (scale bars: 20 μm). b Western blotting analyses on the expression of p-β-catenin (Y142/S552/Y654) in the tissue lysates obtained from a. The protein loading for total β-catenin was normalized and served as a loading control. c Whole-mount co-staining for β-catenin (red) and DAPI (blue) in organoids cultured with/without NGF (scale bars: 50 μm). d Western blotting analyses for the expression of β-catenin in organoids with/without NGF. β-actin served as a loading control. e The level of p-β-catenin (Y142/S552/Y654) in organoids cultured with/without NGF was analyzed by western blotting. The protein loading for total β-catenin was normalized and served as a loading control. f qPCR analyses on the expression of multiple Wnt-target genes, including Lgr5, Sox9, β-catenin, Axin2, Cd44, and Ascl2 in Lgr5^EGFP+ cells isolated from control and NMS mice treated with or without intraperitoneal injection of NGF and MNAC13 (**p < 0.01; ***p < 0.001, n ≥ 5/group; ANOVA). g qPCR analyses for Wnt-responsive genes, including β-catenin, Axin2, Cd44, and Ascl2 in organoids cultured with/without NGF (**p < 0.01; ***p < 0.001, n = 3; two-tailed t test). All data represent the mean ± SEM