Fig. 4

Fig. 4

a Schematic representation of Netrin-1/UNC5B-mediated prosurvival signal. UNC5B domains are indicated with different colors. Blue ovals: Ig-like domains; red ovals: thrombospondin-like domains (TSP1); beige cylinder: transmembrane domain (TM); yellow circle: ZU5 domain; violet cube: UPA/DCC-binding domain (DB); pink circle: death domain (DD). Region encoded by exon 8 is shown in green. Netrin-1 is in orange, whereas DAPk is in dark blue. DAPk phosphorylation at Ser308 is indicated. b Localization of GFP-tagged UNC5B isoforms in HUVECs. Scale bar: 10 μm. c Co-immunoprecipitation of UNC5B AS isoforms GFP-tagged with HA-tagged isoforms in the presence or absence of Netrin-1 (150 ng/ml). Unspecific magnetic beads (Beads) as control. d Vital exclusion assay in HeLa cells overexpressing UNC5B AS isoforms or GFP as control. Error bars indicate ±SD; n = 3. One-way ANOVA for multiple comparisons. e DAPk phosphorylation (Ser308) was evaluated in HeLa cells co-transfected with plasmids expressing DAPk and Unc5b isoforms with or without Netrin-1 treatment (150 ng/ml). Total DAPk as control. f Co-immunoprecipitation of Phospho-DAPk (Ser308) in HeLa cells transiently transfected as in e. g Left: Lateral views (fluorescence) of 72 hpf zebrafish embryos expressing the GFP under the control of the endothelial-specific promoter kdrl injected with a control morpholino (MO-ctr) or with a morpholino against unc5b (MO-unc5b); unc5b morphants were also co-injected with a morpholino-resistant zebrafish mRNA encoding for unc5-Δ8 (unc5b-Δ8) and treated with a pan caspase inhibitor (BAF inhibitor). White arrows: forming PAV. Right: Quantification of PAV formation in the above zebrafish embryos, two different biological replicates were analyzed. n = 2. Error bars indicate ±SEM. Scale bar: 50 μm. h HUVEC/TERT2 treated with a siRNA oligo against the UNC5B-Δ8 mRNA (n  5) or a control (Ctr) oligo (n = 6) were analyzed (left) by RT-PCR for UNC5B exon 8 splicing and (center) for the formation of capillary tube-like structures on Matrigel. Right: quantification of nodes and segments (# per field) in the in vitro tube formation assay. Two-tailed Student’s t-test. Error bars indicate ±SD. n = biologically independent experiments. Exact P values are indicated: *P < 0.05;; ***P < 0.001; ****P < 0.0001; ns not significant.