Knockdown of HOP Expression Does Not Affect the Proliferative Behavior or Apoptotic Rate of Vascular Endothelial Cells We next investigated the molecular mechanism of how HOP is involved in angiogenesis. The angiogenic process requires both the proliferation and migration of vascular endothelial cells [16]. We thus examined whether the depletion of HOP in HUVECs affects their proliferative behavior. HOP shRNA could significantly inhibit the expression of HOP (Figure 2A), but cells transfected with HOP shRNA or the control vector showed very similar growth curves (Figure 2B). This result suggests that knockdown of HOP expression does not affect vascular endothelial cell pro- liferation. Heat shock proteins have been also involved in apoptosis resistance. We thus investigated the effect of HOP depletion on the apoptotic rate of vascular endothelial cells. HUVECs transfected with HOP shRNA or the control vector were stained with fluorescein-conjugated annexin V, which reports the loss of phosphatidylserine asymmetry of the plasma membrane at the early stage of apoptosis [34]. We found that the depletion of HOP does not alter the apoptotic rate of vascular endothelial cells (Figure 2C and 2D). HOP Is Important for Endothelial Cell Migration Migration of endothelial cells is an important step of angiogenesis [35,36]. Therefore, we next wanted to find out whether HOP functions in angiogenesis through regulating endothelial cell migration. HUVECs transfected with HOP or control siRNAs were mechanically scratched to create a wound. After 19 hours, we examined the extent of wound closure. By measuring the wound area compared with the initial wound area, we found that the ability of HUVECs to migrate to the wound HOP Regulates Cell Migration area was significantly inhibited after transfection with HOP siRNAs (Figure 3A and 3B). We further found that overexpression of HOP promoted the migration of HUVECs (Figure 3C and 3D). In addition, HOP shRNA inhibited the effect of wild-type HOP on cell migration, but not the effect of shRNA-resistant mutant HOP on cell migration (Figure 3C and 3D). To verify the role of HOP in cell migration, we performed a transwell migration assay, which was routinely used to study cell migration in response to specific signal stimuli. HUVECs transfected with HOP or control siRNAs were placed in transwell migration chambers containing filters coated with matrigel on the upper side. We examined the number of cells that migrate through matrigel in response to serum stimulation. As shown in Figure 3E and 3F, only a small population of cells transfected with HOP siRNAs could pass through the matrigel, whereas cells transfected with the control siRNA migrated efficiently. These results thus suggest that HOP regulates angiogenesis through promoting endothelial cell migration. HOP Regulates Cell Polarization and Membrane Ruffling in Migrating Cells To further investigate the mechanism by which HOP regulates cell migration, we studied the effect of HOP on cell polarization, a critical step of the cell migration process [36,37]. To examine cell polarization, HUVECs transfected with HOP or control siRNAs were scratched, and fixed 2 hours later for immunoflu- orescent staining. We defined cells with centrosomes localized between the nuclei and the wound edge as polarized cells. As shown in Figure 4A and 4B, nearly 80% of cells in the control group exhibited polarized characteristics. In contrast, cells transfected with HOP siRNAs showed random centrosome localization. Figure 2. Knockdown of HOP expression does not affect the proliferative behavior or apoptotic rate of vascular endothelial cells. A. Immunoblotting analysis of HOP and b-actin expression in HUVECs transfected with HOP shRNA or the control vector for 96 hours. B. Growth curves of HUVECs transfected with HOP shRNA or the control vector, measured by sulforhodamine B staining assay. C. HUVECs transfected with HOP shRNA or the control vector for 48 or 96 hours were stained with fluorescein-conjugated annexin V. Phase contrast and fluorescence images were then taken. D. Experiments were performed as in C, and the percentage of apoptotic cells was then quantified. doi:10.1371/journal.pone.0036389.g002 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e36389