EPZ6438 treatment restored age-related ICC/ICC-SC decline via activation of the SCF-KIT pathway in klotho mice. (A) EPZ6438 (EPZ) restored upregulated H3K27me3 levels (lower left), reduced KIT protein (lower middle), and reduced SCF protein (lower right) in gastric tunica muscularis of klotho mice (n = 7/group). EPZ6438 had no effect on upregulated TRP53 protein (upper left), TRP53 phosphorylation (upper middle), and upregulated EZH2 protein expression (upper right). Vehicle (Veh) was used as a control for EPZ6438. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Reduced gastric ICC networks in klotho mice were restored by EPZ6438 treatment. Representative confocal stacks showing KIT+ (green) and ANO1+ (magenta) myenteric ICC (ICC-MY), and intramuscular ICC (ICC-IM) in corresponding regions of the gastric corpus (B; greater curvature, full thickness) and fundus (C; greater curvature, full thickness) of a WT and klotho mice. n = 5/group. Scale bar: 50 μm. (D) Normalized ICC (KIT+CD34- cells) and ICC-SC (KITlowCD34+ cells) numbers detected by flow cytometry in the nonhematopoietic of gastric muscles of klotho mice (n = 5–6/group). Statistical significance was determined using Kruskal-Wallis 1-way analysis of variance (on ranks). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. ns, not significant.