Figure 5.

Neutralizing the antibody against the integrin receptor reduced omentin-1 binding to the cell membrane. Omentin-1 attenuated the apoptosis and lipid burden induced by ox-low-density lipoprotein (LDL) in macrophages. (A) THP1 cells were induced by 100 ng/ml PMA for 48 h to transform into a macrophage phenotype. After being pretreated by the IgG isotype or neutralizing antibodies against human integrin αvβ3 or αvβ5, the cells were then incubated with Flag-tagged omentin-1. The retention of omentin-1 on the cell membrane was detected by the antibody against the flag tag (PE-labeled). The mean intensity of PE fluorescence was quantified and used to express the magnitude of omentin-1 retention (n = 3). (B) The RAW264.7-derived macrophages were pretreated by omentin-1 (800 ng/ml) for 1.5 h, and then they were co-incubated with high ox-LDL (50 μg/ml) for 24 h. Apoptosis of macrophages was probed by an in situ cell death detection kit (TUNEL), and the apoptotic rate was presented as the percentage of TUNEL-positive nuclei (n = 3). (C) The RAW264.7-derived macrophages were transfected by ITGAV siRNA to knock down the expression of ITGAV. Then they were incubated with omentin-1 and high ox-LDL to induce apoptosis. The apoptotic rate was assessed as described above (n = 3). (D) The RAW264.7-derived macrophages were pretreated by omentin-1 (900 ng/ml) for 1.5 h, and then they were co-incubated with mild ox-LDL (50 μg/ml) for 24 h. The lipid content of the cells was detected by an oil red o stain kit. The integral optical density (IOD) of the oil red o positive area vs. cell counts was calculated to represent the lipid retention in cells (n = 6). (E) The RAW264.7-derived macrophages were transfected by ITGAV siRNA to knock down the expression of ITGAV. Then they were incubated with omentin-1 and mild ox-LDL to induce apoptosis. The lipid retention of cells was assessed as described above (n = 6). All data in this figure were presented as mean ± SEM (n.s., non-significant).