Plexin-A4 modulates bFGF-induced signal transduction. (A) HUVEC expressing sh-control or sh-plexA4 were seeded in gelatin coated 24 well dishes and the number of attached cells determined as described. BFGF (5 ng/mL) was then added and number of attached cells was determined again after 3 days as described. The average of 3 independent experiments each of which was done in triplicates is shown. (B) Incorporation of BrdU into the DNA of HUVECs infected with lentiviruses directing expression of nontargeting shRNA (Sh-Control) or an shRNA targeting plexin-A4 (sh-plexA4) was measured 24 hours after stimulation with bFGF. The number of microscopic fields photographed and counted was 5 and 7 for sh-control, and sh-plexA4, respectively. Shown is a representative experiment of 2 experiments that produced similar results. (C) HUVECs ectopically expressing plexin-A4 or control cells infected with an empty lentiviral expression vector (Control) were seeded in 24 well dishes (2 × 10⁴ cells/well) in the presence or absence of bFGF (5 ng/mL). After 3 days adherent cells were detached and counted in a coulter counter. Shown is one representative experiment of 3 that gave similar results. (D) HUVECs expressing a nontargeting shRNA (sh-control) or HUVECs expressing a plexin-A4 targeting shRNA (sh-plexA4) were stimulated with bFGF (5 ng/mL) or EGF (50 ng/mL). After 10 minutes at room temperature the cells were lysed and ERK1/2 phosphorylation determined using Western blot analysis. Shown is one representative experiment of 3 that gave similar results. (E-F) A full-length human plexin-A4 was fused to a c-terminal V5 tag and expressed along with FGFR-1/VSV (E) or FGFR-2/VSV (F) in PAE cells. The cells were lysed and immunoprecipitation was performed using anti-V5 or anti-HA antibodies used as negative controls. Precipitates were subjected to Western blot analysis using antibodies directed against VSV. Blots were then stripped and reprobed with an anti-V5 antibody. (G) HUVEC expressing sh-plexA4 or sh-control were seeded (1.2 × 10⁴ cells/well) on top of Matrigel. Tube formation and quantification of bifurcations in the tubular network formed were assessed at various time points. Shown is one representative experiment of 3 that gave similar results. (H) Spheroids (500 cells/spheroid) containing HUVECs expressing control or a plexin-A4 targeting shRNA were seeded in collagen and stimulated to sprout with 5 ng/mL bFGF. Shown are representative pictures of sprouting spheroids taken after 24 hours.