HOP Regulates Cell Migration Figure 3. HOP is important for endothelial cell migration. A. HUVECs transfected with HOP or control siRNAs were scratched, and wound margins were imaged 0 or 19 hours later. B. Experiments were performed as in A, and the extent of wound closure was quantified by measuring the wound area compared with the initial wound area. **, p,0.01 versus control. C. HUVECs transfected with GFP, GFP-HOP, GFP-HOP plus HOP shRNA, or GFP-mHOP plus HOP shRNA were scratched, and wound margins were imaged 0, 10, 20, or 24 hours later. D. Experiments were performed as in C, and the extent of wound closure was quantified. *, p,0.05. E. HUVECs transfected with HOP or control siRNAs for 84 hours were placed in transwell migration chambers containing filters coated uniformly on upper side with matrigel. To stimulate migration, 10% FBS was added to the lower chamber. Transwell chambers were stained with crystal violet and imaged after 12 hours. F. Experiments were performed as in E, and the average number of migrated cells was quantified. **, p,0.01 versus control. doi:10.1371/journal.pone.0036389.g003 To better understand the role of HOP in cell polarization, we studied its effect on membrane ruffling at the leading edge, another important feature of migrating cells [38]. HUVECs were transfected with GFP together with HOP shRNA or the control vector, and the fluorescence of GFP at the leading edge of cells was recorded at 20-second intervals to measure membrane ruffle dynamics. We found that HOP shRNA remarkably decreased membrane ruffle dynamics at the leading edge of migrating cells PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e36389