ROS play a role in VL propagation. (Part I) The NADPH oxidase subunit p47phox is enriched in VL. Representative live-cell imaging of p47phox distribution dynamics during diapedesis. MVECs were cotransfected with p47phox-DsRed (red) and either mYFP (green; ex. 1) or actin-GFP (green; ex. 2, corresponding to Fig. 10 a). Example 1 shows leading edge enrichment of p47phox in VL during closure of a paracellular gap. Example 2 shows p47phox coenriched with actin at nodes of VL initiation and in VL leading edge, which propagate successively across a transcellular pore and then a paracellular gap. IRM shown on the far right indicates VL initiate and propagate in close apposition to the substrate. (Part II) Effect of ROS inhibitors on VL-mediated pore closure. Representative live-cell imaging of reversible blockade of VL by ROS inhibitors during diapedesis corresponding to Fig. 10 d. MVECs expressing mYFP were imaged during lymphocyte diapedesis. Where indicated, inhibitors of NAPDH oxidase signaling apocynin (1 mM; ex. 1) or Tempol (500 µM; ex. 2) were added and then at later times washed out (as indicated). In both cases micro-wounds remained patent during the 15-min incubation with drug, and washout led to immediate mobilization of steered VL and micro-wound healing. Images were acquired by time-lapse microscopy using a microscope (Axiovert 200M; Carl Zeiss). Frames were taken every 25 s for 29 min and 5 s (Part I, ex. 1), 20 s for 12 min and 5 s (Part I, ex. 2), 45 s for 46 min and 45 s (Part II, ex. 1), or 45 s for 42 min and 25 s (Part II, ex. 2).
