(A) Quantitation of tube formation by transduced moEC. (B) Transduced moEC were subjected to tube formation assays in the presence of either anti-L1CAM clone 324 or a control, irrelevant antibody (IgG). (C) Quantitation of tube formation by parental moEC treated with CM from moEC transduced either with the empty vector (Vector) or with L1-ΔTM. (D) Quantitation of tube formation assays on moEC untreated or treated with increasing concentrations of recombinant L1-ΔTM. (E) Schematic illustration of the mechanism of action of the morpholino oligonucleotide (L1–SB), which binds to the exon 25/intron 25 junction of L1cam, thus preventing the recruitment of the spliceosome and, hence, impairing the inclusion of exon 25. (F) lu2EC transfected with either an irrelevant morpholino (Ctr) or with L1-SB were analyzed by RT-PCR for the AS of L1cam exon 25 (left, top panel), whereas CM from the same cells were analyzed in immunoblotting with the L1CAM antibody (left, bottom panel). Parental moEC were subjected to tube formation assays in the presence of CM from Ctr- or L1-SB-transfected lu2EC (right panel). (G) Representative images and quantitation of vessel density in matrigel plugs pre-mixed with the CM from ECs transduced with either the empty vector (Vector), L1-FL or L1-ΔTM, and then implanted subcutaneously into C57Bl/6 mice (n = 3 mice/group). Matrigel plugs containing FGF2 served as positive control. Scale bar, 100 μm. Right panel: CD31+ vessels were counted in five different fields. (H) Left panels: immunoblots for phospho-FGFR1 (pFGFR1) and total FGFR1 (FGFR1) on serum-starved moEC left untreated or treated with recombinant L1-ΔTM (20 μg/ml) for 10 or 30 min. The blots were obtained from the same gel, the white line between the blots indicates the removal of intervening lanes. Right panel: FGFR1 phosphorylation in three biological replicates was quantitated by calculating the ratio between phospho-FGFR1 and total FGFR1. Data are normalized against the basal phosphorylation in untreated cells (indicated by the red dashed line). (I) moEC transduced with the empty vector (Vector) or with L1-ΔTM were subjected to tube formation assays in the presence of either the FGFR1 inhibitor PD173074 (PD) or DMSO as a control. For each analysis, data are expressed as means ± SEM from three independent experiments. Comparisons between experimental groups were done with two-sided Student’s t-tests; **p<0.01, ***p<0.001.