LECT2/Tie1/Src signaling regulates the oxidative stress response in endothelial cells and liver ischemia-reperfusion injury

Figure 3 Leukocyte-derived chemotaxin-2/Tie1 signaling promotes Src phosphorylation. A: EA.hy926 cells were subjected to hypoxia-reoxygenation injury after transfection with leukocyte-derived chemotaxin-2 (LECT2)-small interfering RNA. Cell lysates were immunoblotted with indicated antibodies; B: Recombinant LECT2 was added to EA.hy926 cells, and the cell lysates were immunoblotted with the indicated antibodies; C: Recombinant LECT2 and rTie1-Ig3 were simultaneously added to EA.hy926 cells. Cell lysates were immunoblotted with the indicated antibodies; D: Dasatinib and recombinant LECT2 were simultaneously added to EA.hy926 cells. Cell lysates were immunoblotted with the indicated antibodies; E: A cell counting kit-8 assay was used to assess cell proliferation capacity at different time points during reoxygenation after 8 hours of hypoxia. Two-way ANOVA; F: Superoxide dismutase activity was measured at different time points during reoxygenation after 8 hours of hypoxia to assess the level of cellular oxidative stress. Two-way ANOVA; G: Lactate dehydrogenase levels were measured at different time points during reoxygenation after 8 hours of hypoxia to assess cellular cytotoxicity. Data are representative of three independent experiments. One-way ANOVA; H: EA.hy926 cells were subjected to hypoxia-reoxygenation injury and treated with recombinant LECT2 in the presence or absence of dasatinib. After 24 hours, tumor necrosis factor-α mRNA levels were measured. One-way ANOVA. All data are presented as the mean ± SEM. ^aP < 0.05, ^bP < 0.01. rLECT2: Recombinant leukocyte-derived chemotaxin-2; siRNA: Small interfering RNA; PBS: Phosphate buffered saline; H/R: Hypoxia/reoxygenation; TNF-α: Tumor necrosis factor-α; SOD: Superoxide dismutase.