Figure 4. Suppressing MAPK signaling inhibits C59-induced proliferation of intestinal stem cells.

(A) Schematic time line of trametinib, C59, and vehicle (Veh) administration in mice. Trametinib was administered (3 mg/kg, QD) 32 hours prior to administration of vehicle or C59 (100 mg/kg, QD). A total of 4 doses of trametinib and 2 doses of C59 or vehicle were gavaged according to hourly time point (indicated by arrows) prior to euthanasia. (B) The combination of trametinib and C59 treatment blocked proliferation and impaired intestinal homeostasis. EdU was injected 2 hours prior to euthanasia into 4 groups of mice treated with either vehicle or C59 alone, or in combination with trametinib (Tram). Left panels show representative images of total EdU⁺ staining in jejunal crypts. Right panels show higher magnification of crypt as indicated. Scale bar, 25 μm. (C) Top graph shows the number of counts for EdU⁺ cells within 10 cells in the base of the crypt. NS, nonsignificant; *P < 0.05; ***P < 0.001, Kruskal-Wallis 1-way ANOVA test. (D) Trametinib blocks PORCN inhibition–induced burst of proliferation. The number of EdU⁺ cells per 10 cells of the crypt base was determined. C59 treatment increased the number of crypts with 4 or more EdU⁺ cells, and this was reversed by coadministration of trametinib. Vehicle, n = 7. Trametinib plus vehicle, n = 6. C59, n = 8. Trametinib plus C59, n = 6. Error bar indicates SEM; 2 experimental replicates.