Ox ldl

Human monocyte derived from patients with acute monocytic leukemia, specifically THP-1 cell (Procell, Wuhan, China), was used in the study. The chemicals utilized in this study, along with the corresponding companies from which they were purchased, are listed in Supplementary Table S1. The differentiation from THP-1 cell to adherent macrophage was induced by an incubation with 50 nM phorbol 12-myristate 13-acetate (PMA, REF: P8139; Sigma, United States) for 24 h. To synchronize the cells, the differentiated macrophages were cultured in basal RPMI 1640 medium for 24 h and then allocated into the following groups: control, oxLDL, oxLDL + L-Cth 0.1 mM, oxLDL + L-Cth 0.3 mM, and oxLDL + L-Cth 1.0 mM. oxLDL and L-Cth were procured from Zhongshan Golden Bridge and Sigma-Aldrich, respectively. In the oxLDL group, 50 mg/L oxLDL was added and incubated for 6 h. In the oxLDL + L-Cth 0.1 mM group, oxLDL + L-Cth 0.3 mM group, and oxLDL + L-Cth 1.0 mM group, the cells were pre-treated with L-Cth for 30 min and subsequently exposed to 50 mg/L oxLDL for 6 h (Zhu et al., 2014 (link)). Likewise, in the oxLDL + aminooxy acetic acid (AOAA; REF: S4989; Selleck, United States) group and the oxLDL + S-adenosylmethionine (SAM; REF: S910367; Macklin, China) group, the cells were intervened with AOAA or SAM for 30 min, followed by a treatment with 50 mg/L oxLDL for 6 h.

THP-M were exposed to the following conditions: (a) blank (no treatment); (b) oxLDL (THP-M were treated with 25 mg/L oxLDL (Jingke Chemistry, China) for 24 h); (c) IFN-γ (THP-M were treated with 100 ng/mL IFN-γ (Sigma, USA) for 24 h); (d) IFN-γ+oxLDL (THP-M were pretreated with 100 ng/mL IFN-γ for 24 h, then treated with 25 mg/L oxLDL for 24 h); (e) oxLDL+IFN-γ (THP-M were pretreated with 25 mg/L oxLDL for 24 h, then treated with 100 ng/mL IFN-γ for 24 h); (f) IFN-γ+1-MT+oxLDL (THP-M were pretreated with 100 ng/mL IFN-γ for 24 h, then treated with 10 μM 1-MT for 24 h and followed by the treatment of 25 mg/L oxLDL for 24 h); (g) oxLDL+IFN-γ+1-MT (THP-M were pretreated with 25 mg/L oxLDL for 24 h, then treated with 100 ng/mL IFN-γ for 24 h and followed by treatment with 10 μM 1-MT for 24 h). (h) IFN-γ+Incyte (INCB024360)+oxLDL (THP-M were pretreated with 100 ng/mL IFN-γ for 24 h, then treated with 20 nM Incyte for 24 h and followed by treatment with 25 mg/L oxLDL for 24 h); (i) oxLDL+IFN-γ+Incyte (THP-M were pretreated with 25 mg/L oxLDL for 24 h, then treated with 100 ng/mL IFN-γ for 24 h and followed by the treatment of 20 nM Incyte for 24 h).

The differentiated THP-1 macrophages were incubated using 10, 30, 50, or 70 μg/mL ox-LDL (Sigma, USA) in an incubator (temperature: 37°C, saturated humidity: 5% CO₂) for 24 h to determine the appropriate ox-LDL concentration (19 (link)). The transfected THP-1 macrophages were then treated with 50 μg/mL of ox-LDL (Sigma) for 24 h.