Ox ldl

Human umbilical vein endothelial cells (HUVECs), and human monocyte-like cell line (THP-1) were cultured in HUVEC specific medium (PROCELL, CHINA, Ham's F-12 K, 0.1 mg/mL Heparin, 0.03–0.05 mg/mL ECGs, 1% P/S) or RPMI-1640 (Gibco, USA) medium supplied with 10% fetal bovine serum (Gibco, USA). The THP-1 cells were stimulated with phorbol ester (PMA) (100 nM, sigma, USA) to differentiate macrophage-like sticky cells for later experiments. Peritoneal macrophages were isolated as previously described [14 ]. Human peripheral blood sample from healthy volunteers (HV) or patients with coronary artery disease (CAD) were obtained from the Ningbo First Hospital, which was allowed by the ethics committee of Ningbo First Hospital. Human peripheral blood-derived mononuclear cells, separated from human peripheral blood with human lymphocyte separation medium (Sigma), were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and then were differentiated into macrophages in the presence of 50 ng/ml of recombinant human GM-CSF (Peprotech) for 4 days, as previously described [17 –19 (link)]. IFN-γ was purchased from BioLegend. ox-LDL and recombinant human TNF-α protein were purchased from Beyotime.

A commercial kit for β-galactosidase (β-gal) staining was used to assess the RAW264.7 senescence induced by ox-LDL (Beyotime Biotechnology, China). For cell senescence assay, ctrl shRNA or caveolin-1 shRNA transfected Raw264.7 cells were treated with oxLDL or left untreated. Twenty-four hours later, cells were washed three times with PBS when grown up to 80% confluence. The fixation solution which included 2% formaldehyde, 0.2% glutaraldehyde was added to cells at room temperature. After 5 min, cells were washed three times with PBS. Then, freshly prepared 1 ml SA-β-gal was used to stain the cells at 37°C for 12–16 h. The blue precipitates can be seen in the cytoplasm. For each sample, six fields were randomly selected. The percentage of positive cells was calculated.