Panel A: A vertical bar graph shows the endothelial cortactin mean fluorescence intensity (MFI) in HUVECs with neutrophil serine protease (NSP) inhibition. The x-axis represents different treatments (DMSO, Sivelestat (Siv) at 50 micromolar and 100 micromolar, PMSF at 0.5 millimolar and 1 millimolar) and the y-axis represents the MFI. The graph indicates that PMSF significantly reduces cortactin degradation compared to DMSO and Sivelestat. Panel B: Another vertical bar graph shows endothelial cortactin MFI with neutrophils treated with medium, PMSF at 2 millimolar, and AEBSF at 0.5 millimolar. The x-axis represents these treatments and the y-axis represents the MFI. The graph shows that both PMSF and AEBSF significantly reduce cortactin degradation. Panel C: A western blot image shows the levels of VE-Cadherin, cortactin, GAPDH, and Cathepsin G (CatG) in HUVEC lysates incubated with 1 or 2 microgram CatG for 20 minutes on ice. Panel D: Another western blot image shows the levels of VE-Cadherin, vinculin, cortactin, gamma-tubulin, GAPDH, and CatG in HUVEC monolayers incubated with 10 µg/mL CatG for 1 hour before washing and protein extraction. Panel E: A vertical bar graph shows the endothelial cortactin levels in TNF-treated HUVEC monolayers incubated with cell-free neutrophil supernatant (cf-NS) or medium alone for 1 hour. The x-axis represents these treatments and the y-axis represents the cortactin levels. The graph indicates a significant reduction in cortactin levels with cf-NS treatment. Panel F: Another vertical bar graph shows endothelial CatG levels under the same conditions as Panel E. The x-axis represents the treatments and the y-axis represents the CatG levels. The graph shows a significant increase in CatG levels with cf-NS treatment. Panel G: A western blot image shows the levels of actin-related proteins (cortactin, vinculin, Arpin, ArpC5a, GAPDH, and CatG) in HUVEC monolayers after co-culture with neutrophils or cf-NS, with or without AAT treatment. Panel H: An overexposed western blot image shows CatG levels in HUVEC monolayers incubated with cf-NS. Panel I: A vertical bar graph shows the endothelial cortactin MFI in HUVEC and neutrophil co-cultures under flow conditions, with permeabilized (Perm positive) or non-permeabilized conditions (Perm-). The x-axis represents these conditions and the y-axis represents the MFI. The graph indicates significant differences in cortactin levels under different conditions. Panel J: Another vertical bar graph shows endothelial CatG MFI under the same conditions as Panel I. The x-axis represents the conditions and the y-axis represents the MFI. The graph shows significant differences in CatG levels under different conditions.
Endothelial cortactin is degraded by NSP. (A and B) Flow cytometric quantification of cortactin MFI in HUVEC with NSP inhibition. Neutrophils (1 × 106/ml) were treated with the NE inhibitor Siv, the general serine protease inhibitors PMSF and AEBSF, and vehicle (DMSO or RPMI medium) for 30 min before co-incubation with activated HUVEC monolayers for 15 min. HUVEC without neutrophils (−) were used as control of basal cortactin levels (n = 3–5 independent experiments). (C) Western blot (VE-Cadherin, cortactin, GAPDH, and CatG) from HUVEC lysates incubated with 1 or 2 μg CatG for 20 min on ice (n = 3). (D) Western blot (VE-Cadherin, vinculin, cortactin, γ-tubulin, GAPDH, and CatG) from HUVEC monolayers incubated with 10 μg/ml CatG for 1 h before extensive washing and protein extraction (n = 3). (E and F) Flow cytometric quantification of endothelial cortactin (E) and CatG (F) levels from TNFα-treated HUVEC monolayers that were incubated with cell-free neutrophil supernatant (cf-NS) or medium alone for 1 h (n = 3–5 independent experiments). (G) Western blot of actin-related proteins from HUVEC monolayers after co-culture with neutrophils or cf-NS and with or without AAT treatment (10 μg/ml) (n = 3). (H) Overexposed western blot from HUVEC monolayers incubated with cf-NS to detect CatG (n = 3). (I and J) Flow cytometric quantification of endothelial cortactin (I) and CatG (J) MFI from HUVEC and neutrophil co-cultures under flow conditions. Flow cytometry was performed under permeabilized (Perm+) or non-permeabilized conditions (Perm−) (n = 3–5). Data are represented as means ± SEM; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant. Source data are available for this figure: SourceData F3.