CYLD modulates microtubule dynamics and cell polarization in migrating cells. (A) HUVECs were transfected with pEGFPC1–α-tubulin and pSUPER or pSUPER-CYLD, and the dynamics of individual GFP-labeled microtubules in migrating cells were then recorded with the TCS SP5 confocal microscope (Leica), equipped with a live-cell imaging workstation and the LAS AF software. Objective lens used was HCX Plan-Apochromat 63×/1.4 NA oil (Leica). (B) Experiments were performed as in panel A, and individual microtubule life history plots representing changes in microtubule length over time were generated. Growth events exhibit an increase in distance from a fixed point (y-axis) over time (x-axis), and shortening events are seen as a decrease in distance over time. (C) HUVECs transfected with CYLD or control siRNAs were scratched, and cells were fixed 2 hours later and stained with anti–α-tubulin antibody, anti–pericentrin antibody, and DAPI to visualize microtubules (green), centrosomes (red), and nuclei (blue), respectively. Broken white lines indicate the wound direction. Objective lens used was Plan-Apochromat 63×/1.25 NA oil (Carl Zeiss Inc).