Fig. 1

KAI1 was predominantly expressed in PCs rather than in ECs, and KAI1 expressed in PC has anti-angiogenic effects. a Left, retinal vasculature of the wild-type (WT) and Kai1 knockout (Kai1−/−) mice (postnatal day 2 and 5 days of age). Endothelial cells were stained with BS-I lectin. Scale bar, 100 μm. Right, quantification of the obtained results (Means ± SEM, 4–9 technical replicates). b Left, filopodia of retinal vessels in WT and Kai1−/− mice (postnatal day 4). Scale bar, 20 μm. Right, quantification of the obtained results (*p < 0.01, **p < 0.05, Means ± SEM, 3 or 4 technical replicates). c Tracking of Kai1 expressing cells in retina of the Kai1-GFP fusion knock-in mice. Confocal images of isolectin B4 (IB4, white), NG2 (red). Right, high-magnification images of the boxed area in the left panel Kai1 (green) was expressed in NG2 positive PCs (Arrow), but not ECs (Arrowhead). Asterisk indicates Kai1-expressing leukocyte or erythrocyte in vessel. Scale bar, 5 μm. d Kai1 mRNA expression in mouse primary ECs and cell lines (MS1, endothelial cell line), and mouse primary PCs and cell line (10T1/2, pericyte cell line). **p < 0.05. e KAI1 protein expression in the mouse primary ECs, mouse primary PCs, human cell lines, HUVECs and HBVPs. *p < 0.01. f Left, in vitro co-culture matrigel tube formation assay using GFP expressing mouse ECs (MS1) co-cultured with Kai1^−/− or WT mouse primary PCs. Scale bar, 100 μm. MS1 cells were transduced by lentivirus encoding GFP. Right, quantification of the obtained results (Means ± SEM, 7 technical replicates). g Left, In vitro matrigel tube formation assay, performed using co-culture spheroids, consisting of primary ECs and PCs, transduced with Myc-Kai1 vector and observed using time-lapse microscopy at the indicated time, 0 and 107 h. Scale bar, 50 μm. Right, quantification of the obtained results (***p < 0.001, Means ± SEM, 3 technical replicates). h Left, in vitro tube formation assay in MS1 and 10T1/2 co-cultured cells, transduced with the indicated adenovirus. Scale bar, 200 μm. Right, quantification of the obtained results (*p < 0.01, **p < 0.05, ***p < 0.001, Means ± SEM, 4 technical replicates)