Bosutinib and nilotinib can attenuate Thr-induced barrier disruption in HBMECs. (A) Left, schematic overview. An xCELLigence assay was used to measure thrombin (Thr)-induced barrier disruption in an HBMEC monolayer. Traces were normalized to media control. KIs (0.5 μM) were added 7 minutes after Thr to assess barrier protective activity. Right: the total change in the cell index is summarized as the area under the curve (AUC) quantification relative to that of DMSO treatment (100%). The data are presented as mean ± standard deviation (SD; n = 3, each done in duplicate). (B) Dose titration of bosutinib, nilotinib, and imatinib in the xCELLigence assay in Thr-treated HBMECs. The data are presented as mean ± SD (n = 5, each done in triplicate). (C) Top, quantification of Thr-induced barrier permeability at the maximum disruption time point (25 minutes) and at a mid-recovery time point (50 minutes), determined using xCELLigence assay. The data are presented as means ± SD (n = 3, each done in duplicate). Bottom left, representative confocal images of HBMECs under resting or Thr-activated conditions at 50 minutes in the presence or absence of KIs (0.5 μM). The fluorescence of VE-cadherin (green), phalloidin (red), and DAPI (blue) are shown. Gaps are indicated as white arrowheads. Bottom right panel: the highlighted blue areas represent gaps in the monolayer. ImageJ quantification showing the percentage intercellular gaps per field, calculated from 8 uniformly sampled fields (n = 3). A 1-way analysis of variance (ANOVA), followed by Dunnett multiple comparison test, was used to analyze the data. ∗P < .05; ∗∗∗∗P < .0001.