Silencing type-A plexins in HUVECs with shRNAs induces plexin specific morphologic effects. (A) The expression levels of the mRNAs encoding type-A plexins were determined in HUVECs infected with nontargeting shRNA (sh-control) and in HUVECs in which the expression of plexin-A1 (sh-plexA1), plexin-A3 (sh-plexA3), or plexin-A4 (sh-plexA4) was silenced with specific shRNAs using real-time quantitative PCR (left panel). Neuropilin levels were also determined in cell lysates using Western blot analysis (right panel). (B) HUVECs expressing sh-control or different sh-plexins as indicated were stained with fluorescent phalloidin as described in “Immunocytochemistry” and photographed. (C-D) The percentage of the HUVECs which changed their morphology after the silencing of Plexin-A1 or Plexin-A4 was determined. Cells in 6 microscopic fields (∼ 300 cells) were photographed and scored using a phase contrast microscope at 20× magnification.