14 15 eeze

Rabbit anti-human antibodies against integrin αv and β3, FAK, p-FAK, PI3K, p-PI3K, AKT, p-AKT, E-cadherin, N-cadherin, vimentin, snail and LY294002 (PI3K inhibitor) were purchased from Cell Signaling (Danvers, MA, USA). slug and PF562271 were purchased from Abcam (Cambridge, MA, US). The small interfering RNAs (si RNAs) against integrin αv, integrin β3 and control for experiments using targeted siRNA transfection were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The 14, 15-EET and 14, 15-EEZE were purchased from Cayman chemical (Ann 152 Arbor, MI, USA).

Cells were transferred to 6-well plates after passaging and incubated in a 37 °C normoxic incubator (5% CO₂, 95% air, 95% humidity). When the monolayer cells grew to 80% confluence, 50 ng/ml TNF-α was added to simulate the endothelial cell injury model. 4 h later, the medium was removed and the cells were washed twice with phosphate buffer (PBS). Then the cells were pretreated with 14,15-EEZE (10 μmol/l, Cayman Chemicals), a selective EET antagonist [20 (link)], for 1 h. After that, 14,15-EETs (1 μmol/l, Sigma) or lysis medium (DMSO) were added to the culture medium. 1 h later, anoxia/reoxygenation (AR) was performed. The cell culture medium was replaced with a sugar-free serum-free medium and incubated in a 37 °C anoxic incubator (5% CO₂, 95% nitrogen). 8 h later, the cells were replaced with a complete cell culture medium and incubated in a normoxic incubator for 16 h. When the cell growth reached 80% confluence in the normoxic group, the corresponding reagents were added and incubation was continued in the normoxic incubator for 24 h without AR.