Figure 4.
Bosutinib can attenuate parasite-induced barrier disruption in HBMECs. (A) Schematic overview of the parasite-permeability assay. Representative xCELLigence recordings of HBMECs treated with lysates from 3D7 Pf-IEs. The traces were normalized to that of uninfected RBC lysates. KIs (0.5 μM) were added at the same time as the parasite lysate. Right, total change in cell index is summarized as the AUC relative to that of DMSO (100%). The data are presented as the mean ± SD (n = 4, done in duplicate). ∗P < .05 determined using a 1-way ANOVA, followed by Dunnett multiple comparison test (compared with the DMSO/Pf-IEs lysate group). (B) 3D7 Pf-IE lysate–induced phosphorylation of VE-cadherin (Y685) and paxillin (Y118) were probed using immunoblotting, along with their respective total proteins. GAPDH was used as a loading control. The western blot images are representative of 3 independent biologic replicates. (C) Upper panel, the 3D7 Pf lysate–induced fold change of the indicated phosphoproteins levels, adjusted to the total protein amounts (dashed lines), is plotted above the Pf lysate-induced change in the cell index (solid lines), measured using the xCELLigence platform in panel (A). Fold change was calculated by normalizing the data against the data of the resting state (nontreated, media only). Lower panel, The AUC was calculated for the temporal phosphoprotein kinetics (total protein normalized) in the presence of KIs or for the DMSO control (100%). Bars represent the mean AUC ± SD (n = 3). ∗∗∗P < .001; ∗P < .05 determined using 1-way ANOVA, followed by Dunnett multiple comparison test (compared with DMSO control). (D) Upper panel, representative recording of xCELLigence barrier response to schizont-stage IT4var19-IEs (after normalization to uninfected RBCs). DMSO or KIs (0.5 μM) were added after 1 hour of co-culture of purified schizont-stage IEs or RBCs with HBMECs. Lower panel, quantification of barrier protective activity of KIs against schizont-induced barrier disruption. The data are presented as mean ± SD (n = 4, each done in triplicate). ∗∗∗P < .001 determined using a 1-way ANOVA, followed by Dunnett multiple comparison test (compared with the DMSO/Pf-IEs group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pVE-Cadh, protein VE-cadherin; RBCs, red blood cells.

Bosutinib can attenuate parasite-induced barrier disruption in HBMECs. (A) Schematic overview of the parasite-permeability assay. Representative xCELLigence recordings of HBMECs treated with lysates from 3D7 Pf-IEs. The traces were normalized to that of uninfected RBC lysates. KIs (0.5 μM) were added at the same time as the parasite lysate. Right, total change in cell index is summarized as the AUC relative to that of DMSO (100%). The data are presented as the mean ± SD (n = 4, done in duplicate). ∗P < .05 determined using a 1-way ANOVA, followed by Dunnett multiple comparison test (compared with the DMSO/Pf-IEs lysate group). (B) 3D7 Pf-IE lysate–induced phosphorylation of VE-cadherin (Y685) and paxillin (Y118) were probed using immunoblotting, along with their respective total proteins. GAPDH was used as a loading control. The western blot images are representative of 3 independent biologic replicates. (C) Upper panel, the 3D7 Pf lysate–induced fold change of the indicated phosphoproteins levels, adjusted to the total protein amounts (dashed lines), is plotted above the Pf lysate-induced change in the cell index (solid lines), measured using the xCELLigence platform in panel (A). Fold change was calculated by normalizing the data against the data of the resting state (nontreated, media only). Lower panel, The AUC was calculated for the temporal phosphoprotein kinetics (total protein normalized) in the presence of KIs or for the DMSO control (100%). Bars represent the mean AUC ± SD (n = 3). ∗∗∗P < .001; ∗P < .05 determined using 1-way ANOVA, followed by Dunnett multiple comparison test (compared with DMSO control). (D) Upper panel, representative recording of xCELLigence barrier response to schizont-stage IT4var19-IEs (after normalization to uninfected RBCs). DMSO or KIs (0.5 μM) were added after 1 hour of co-culture of purified schizont-stage IEs or RBCs with HBMECs. Lower panel, quantification of barrier protective activity of KIs against schizont-induced barrier disruption. The data are presented as mean ± SD (n = 4, each done in triplicate). ∗∗∗P < .001 determined using a 1-way ANOVA, followed by Dunnett multiple comparison test (compared with the DMSO/Pf-IEs group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pVE-Cadh, protein VE-cadherin; RBCs, red blood cells.

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