Fig. 1.

Effects of KIF and AUY-922 on lung endothelial cells.

Western Blot analysis of (A) phosphorylated Cofilin (pCofilin) and Cofilin (B) phosphorylated MLC2 (pMLC2) and MLC2 after 24 h treatment of BPAEC with either KIF (5 μM) or vehicle (0.1% DMSO) and post-treatment with AUY-922 (2 μM) or vehicle (0.1% DMSO) for 16 h. The blots shown are representative of 4 independent experiments. The signal intensity of the bands was analyzed by densitometry. Protein levels of pCofilin and pMLC2 were normalized to Cofilin and MLC2 respectively. *P < 0.05, **P < 0.01 vs vehicle. Means ± SEM. Western Blot analysis of (C) phosphorylated MLC2 (pMLC2) and MLC2 after 24 h treatment of HuLEC with either KIF (5 μM) or vehicle (0.1% DMSO); and post-treatment with AUY-922 (2 μM) or vehicle (0.1% DMSO) for 16 h. The blots shown are representative of 3 independent experiments. The signal intensity of the pMLC2 was analyzed by densitometry. Protein levels of pMLC2 were normalized to MLC2. *P < 0.05 vs vehicle. Means ± SEM. (D) Confluent monolayers of BPAEC were pre-treated with either vehicle (0.1% DMSO) or KIF (25 μM) (red arrow) for 18 h, followed by treatment with either vehicle (0.1% DMSO) or 5 μM of AUY-922 (green arrow). A gradual increase in endothelial permeability (reduced TEER) was observed in KIF treated cells (red line). AUY-922 significantly reduced the endothelial permeability (increased TEER) in both KIF-pretreated (blue line) and vehicle-treated cells (green line). (E) Cells were incubated with either VEH (0.1% DMSO) or KIF (0.01, 0.1, 1, 10, 100, 200 μM) or AUY-922 (0.01, 0.1, 1, 10, 100, 200 μM) for 24 h. Cellular viability was evaluated by employing the MTT assay. ***P < 0.001 vs VEH, n = 3. Means ± SEM. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)