Fig. 5: Mice with IGF-1-deficient macrophages have decreased intestinal endothelial cell proliferation.

a Breeding scheme for generating mice with IGF-1 sufficient and IGF-1-deficient macrophages. b, c IGF-1 protein is decreased in isolated CX3CR1⁺ cells from pups with IGF-deficient macrophages (Igf-1^ΔMϕ) compared with IGF-1-sufficient littermates (Igf-1^f/f). For each group and in each experiment, intestines from 8 to 10 pups were pooled together to obtain enough cells for western blot analysis. b Western blot image of one experiment. c The experiment was repeated twice, and band density was quantified (mean ± SEM, n = 3 /group). d, e Tissue sections from 24-hour-old Igf-1^ΔMϕ pups (n = 6) or IGF-1-sufficient pups (n = 8) were stained for Ki-67 (green) and endomucin (red). d Typical image is shown, scale bar: 50 μm. e Average proliferating endothelial cells (Ki-67⁺ endomucin⁺) per ×20 field in the intestinal villi (see arrows in d) were counted from 3 to 4 field images per sample. Each dot indicates the number from one image. f–h Isolated small intestinal LP cells from D0, D1, D4, D20 IGF-1-sufficient or Igf-1^ΔMϕ pups were gated on single/live CD31⁺CD45⁻ cells and analyzed for CD31 and Ki-67. f Typical flow cytometry images are shown. g The graph represents the percentage of proliferating (Ki-67⁺) endothelial cells at different ages in the intestine of IGF-1-sufficient or Igf-1^ΔMϕ pups (mean ± SEM). h Endothelial cell number relative to the isolated total cell number per intestine, which is subsequently normalized to Igf-1^f/f group, is shown here (mean ± SEM), n = 6–9/group (g, h, see dots in panels and Source Data file for exact n number). P values were calculated using two-sided Student’s t tests (c, e) or multiple t tests (g, h). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.