Fig. 7

UXT potentiates angiogenesis through Notch signaling. (A-C) UXT potentiates angiogenesis in vitro. (A) Knocking down UXT by shRNAs or ectopically expressing wild-type or mutant UXT (phage plasmids) was performed in HUVECs, followed by the in vitro angiogenesis assay. Fluorescence images were taken (A) and quantified (B,C). Scale bar: 500µm. Relative mean mesh area (B) and relative branching length (C) were calculated by using Image J. (D-F) 3D in vitro angiogenesis with collagen gel-embedded cytodex beads coated with HUVECs, treated with shNT, shUXT, phage-vector or phage-UXT. Cumulative length and the numbers of all sprouts originating from an individual cytodex bead were quantified using ImageJ after 24h, with ten cytodex beads analyzed per experimental group. Scale bar: 200 µm. (G-I) Fluorescence images (G) and quantification (H,I) of the in vitro angiogenesis assay on HUVECs treated with DMSO or DAPT. Wild-type (shNT) or UXT-deficient (shUXT) HUVECs were seeded on Matrigel and then treated with either DMSO or DAPT (1.5µM) for 6h. Scale bar: 500µm. Relative mean mesh area (H) and relative branching length (I) were calculated using ImageJ. The data were normalized on the basis of the corresponding input control. (J-Q) Tg(kdrl:EGFP)^s843 embryos were treated with either 60µM DAPT or DMSO. (J,K) Tg(kdrl:EGFP)^s843 embryos injected with 4ng of control morpholino (Ctrl MO) were treated with DMSO. (N,O) Tg(kdrl:EGFP)^s843 embryos injected with 4ng of control morpholino were treated with DAPT. (L,M) Tg(kdrl:EGFP)^s843 embryos injected with 4ng of UXT morpholino (UXT MO) were treated with DMSO. (P,Q) Tg(kdrl:EGFP)^s843 embryos injected with 4ng of UXT morpholino were treated with DAPT. All images were taken at 30hpf. The confocal images in K,O,M,Q show higher magnification of areas of the bright-field images to the left. The red arrowheads indicate the robust angiogenesis in ISVs, whereas the yellow arrowheads highlight the DLAV growth rescued by DAPT treatment. Scale bar: 100µm. (R-U) 2ng of RBP-J_K morpholino were injected into Tg(kdrl:EGFP)^s843 embryos without (R,S) or with (T,U) 4ng of UXT morpholino. All the images were taken at 30hpf. The confocal images in S,U show higher magnification of areas of the bright-field images to the left. Red arrowheads indicate excessive angiogenesis in ISVs, and yellow arrowheads denote DLAV growth rescued by co-injection of the RBP-J_K morpholino. Scale bar: 100µm. (V) Quantification of J-U. The ISV branching points per segment were analyzed. Five different embryos were analyzed per experimental group. The data were normalized on the basis of the corresponding input control. All quantitative data are presented as the mean±s.e.m. (at least three independent experiments); *P<0.05, **P<0.01, ***P<0.001 versus the corresponding control.