Fig 1: LONG R3rhIGF1 treatment restored age-related ICC/ICC-SC decline via activation of the ERK1/2 pathway in klotho mice. (A) LONG R3 rhIGF1 (IGF1) restored the decline in ERK phosphorylation (upper left panel), KIT protein (upper middle panel), and ETV1 protein (upper right panel), and mitigated increased DNA damage response-associated ?-H2A.X (H2AXS139p) protein (lower left panel), increased CDKN1B protein (lower middle left panel), reduced CCND1 protein (lower middle right panel), and reduced KITL protein (lower right panel) in gastric tunica muscularis of klotho mice (n = 8–11/group). ACTB (actin beta) was used as a loading control. (B) Reduced gastric ICC networks in klotho mice were restored by LONG R3 rhIGF1 treatment. Representative confocal stacks showing KIT+ (magenta) and ANO1+ (green) ICC in corresponding regions of the gastric corpus (greater curvature, full thickness) of a WT and klotho mice. N = 3/group. Scale bar: 10 µm. (C) Restored ICC (KIT+CD34- cells) and ICC-SC (KITlowCD34+ cells) numbers detected by flow cytometry in the nonhematopoietic CD44+ fraction of gastric muscles of klotho mice (n = 15/group). Statistical significance was determined using Kruskal–Wallis 1-way analysis of variance (ANOVA on ranks). HP, hematopoietic cells; P-ERK1, Pextracellular signal-regulated kinase 1; T-ERK1, T-extracellular signal-regulated kinase 1.