Inhibition of sema6B expression in HUVECs mimics the effects of plexin-A4 silencing. (A) HUVECs were seeded and after 4 hours the medium was exchanged with conditioned medium derived from HEK293 cells transfected with empty expression vector (control) or conditioned medium from HEK293 cells expressing the sema6A extracellular domain tagged with an Fc tag (sema6A-Fc). bFGF (5ng/mL) was then added or not. The number of adherent cells was then determined as described. The average of 2 independent experiments each of which was done in triplicates is shown. (B) The effect of sema6A-Fc on the survival of HUVECs expressing the sh-control or sh-plexA4 was determined as described without the stimulation of bFGF. (C) The expression of sema6B mRNA in HUVECs infected with lentiviruses expressing control shRNA or a sema6B targeting shRNA was determined using real time PCR (left). The actin cytoskeleton of control and sema6B silenced cells was visualized with fluorescent phalloidin (right). (D) HUVECs expressing control shRNA or sema6B shRNA were seeded in 24 well dishes in the presence or absence of bFGF (5 ng/mL). After 3 days adherent cells were detached and counted. Hundred percent represents the number of adherent cells/well as counted 4 hours after seeding. The average of 2 independent experiments each of which was done in triplicates is shown. (E) HUVECs expressing sh-control or sh-sema6B were stimulated with bFGF (5 ng/mL) for 10 minutes at room temperature. The cells were lysed and ERK1/2 phosphorylation was determined using Western blot analysis. Shown is a representative experiment of 3 performed with similar results. (F) Expression of sema6B was determined in western blots prepared from cell lysates of BHK-21 cells expressing a control plasmid or cells over-expressing myc-tagged sema6B using an antibody directed against myc. (G) Expression of plexin-A4 was determined in western blots prepared from cell lysates of parental BHK-21 cells or cells over-expressing recombinant plexin-A4 using an antibody directed against plexin-A4. (H) BHK-21 cells over-expressing recombinant sema6B or control cells infected with an empty expression vector were seeded in triplicates in 24-well dishes (2 × 10⁴ cells/well) in the presence or absence of increasing concentrations of bFGF. After 3 days adherent cells were detached and counted in a coulter counter. Shown is a representative experiment of 3 performed with similar results.