VL are enriched in and functionally dependent on NADPH oxidase activity. (a) MVECs coexpressing p47phox-DsRed and actin-GFP were imaged live during T cell diapedesis. p47phox was enriched in ventral (IRM) actin-rich initiation nodes (N) and in VL leading edge (arrowheads). See Video 10. (b) Representative ratiometric images of H₂O₂ production in (i) pore- and (ii) gap-closing VL in MVECs expressing the biosensor mHyPer during T cell diapedesis. (c) Quantification of p47phox (i) and H₂O₂ (ii) enrichment in VL. n > 13. (d, i) Representative NADPH oxidase inhibition/washout imaging experiment. After multiple T cell diapedesis events on MVECs expressing mYFP, apocynin was added. Arrows indicate multiple pores and gaps that persisted for 15 min, but were rapidly healed by steered VL (arrowheads) after drug washout. See Video 10. Experiments as in d (i) were performed with VAS-2870, DPI, PEG-catalase, and DMSO (control) and quantified by cumulative wound closure analysis, shown as a continuous line plot (ii) and bar graph of single pre/post-washout time points (iii). n > 5. (e) MVECs coexpressing mDsRed and mHyPer were imaged during probe wounds and after addition of VAS-2870. Representative mDsRed images (i), kymograph (ii), mHyper ratiometic images (iii), and quantitation (iv) are shown. n = 3. Values represent mean ± SEM. Statistical significance is indicated with p-values as follows: ***, P < 0.001; *, P < 0.05. Bars, 5 µm.