Figure 1: Endothelial CTR1 is required for postnatal angiogenesis in vivo.

A and B. Retinal whole-mount staining with Isolectin B4 (IB4) of P6 WT and CTR1^iECKO mice. Arrowheads show tip cell sprouting and filopodia (A). BrdU or ERG (endothelial nuclei marker)(green) co-stained with or without IB4 (red) in P6 retinal flatmounts of WT and CTR1^iECKO mice (B). Right panels show quantification of vascular progression length, numbers of branch point, tip cells and filopodia (A) and BrdU -or ERG-positive cells or endothelial BrdU-positive cells in the field (B), (vascular progression: n=11 samples each for WT and CTR1^iECKO; branch point, tip cells, filopodia, BrdU+, ERG+: n=6 samples each for WT and CTR1^iECKO respectively, compared with two-tailed unpaired t-test). C. Blood flow recovery after hindlimb ischemia as determined by the ratio of foot perfusion between ischemic (left) and non-ischemic (right) legs in WT and CTR1^iECKO mice (right), two-way ANOVA followed by Bonferroni’s multiple comparison analysis. Left panels show representative laser Doppler images of legs at day 14. Bottom panels show CD31⁺ staining (capillary density) in ischemic and nonischemic gastrocnemius muscles at day 14. Right panel shows quantification, (n=6 mice per group, representative of two independent experiments, compared with two-tailed unpaired t-test). D. Excisional cutaneous wounds were created using a 3 mm biopsy punch on the dorsal skin of WT and CTR1^iECKO mice. Ruler notches=1 mm. Right panel shows the quantification of wound closure rates, two-way ANOVA followed by Bonferroni’s multiple comparison analysis. Bottom images show CD31 staining of wounded tissues at day 7 with magnified images in Box. Right panel shows quantification, (n=6 mice per group, representative of two independent experiments, compared with two-tailed unpaired t-test). E. Adenovirus encoding VEGF164 (1×10⁹ pfu) (Ad-VEGF) or β-galactosidase (Ad-LacZ) was intradermally injected into the right and left ear skin of WT and CTR1^iECKO mice, respectively. Ear vasculature (red) was visualized by a whole-mount staining with CD31. Right panel shows the quantification of vessel density. (n=5 mice per group, representative of two independent experiments, two-tailed unpaired t-test. *p<0.05, **p<0.005, ***p<0.001. Data are mean ± SEM). Source numerical data are available in source data.