Research Paper Volume 13, Issue 18 pp 22412—22431

Pim-2 kinase inhibits inflammation by suppressing the mTORC1 pathway in atherosclerosis

Ox-LDL upregulates the expression of Pim-2, the mTOR pathway and inflammatory cytokines in THP-1-derived macrophages. THP-1-derived macrophages were sequentially treated with 50 μg/ml of ox-LDL for 12 h, 24 h, 36 h, and 48 h. (A) Representative western blot analysis of Pim-2, p-mTOR (Ser2448) and mTOR, p-S6K1 (Thr389) and S6K1, and p-4EBP1 (Thr37/46) and 4EBP1. β-Actin was used as a loading control. (B, C) Corresponding densitometric analysis of blots in (A). (D) Intracellular lipid droplets were stained with oil red O working solution in THP-1-derived macrophages treated with 50 μg/ml of ox-LDL for 24 h. (E) Quantification of positive staining for lipid droplets (n=3 per group). (F) mRNA expression of inflammatory cytokines, including IL-6, MCP-1, TLR-4 and TNF-α, was determined by quantitative RT-PCR in ox-LDL-treated THP-1-derived macrophages; values normalized to the housekeeping gene GAPDH. (G) The concentrations of TC, FC, and CE were determined using the TC/FC Quantification Assay (n=3 per group). The data are represented as the means ± SD of three independent experiments; *P P P

Figure 1. Ox-LDL upregulates the expression of Pim-2, the mTOR pathway and inflammatory cytokines in THP-1-derived macrophages. THP-1-derived macrophages were sequentially treated with 50 μg/ml of ox-LDL for 12 h, 24 h, 36 h, and 48 h. (A) Representative western blot analysis of Pim-2, p-mTOR (Ser2448) and mTOR, p-S6K1 (Thr389) and S6K1, and p-4EBP1 (Thr37/46) and 4EBP1. β-Actin was used as a loading control. (B, C) Corresponding densitometric analysis of blots in (A). (D) Intracellular lipid droplets were stained with oil red O working solution in THP-1-derived macrophages treated with 50 μg/ml of ox-LDL for 24 h. (E) Quantification of positive staining for lipid droplets (n=3 per group). (F) mRNA expression of inflammatory cytokines, including IL-6, MCP-1, TLR-4 and TNF-α, was determined by quantitative RT-PCR in ox-LDL-treated THP-1-derived macrophages; values normalized to the housekeeping gene GAPDH. (G) The concentrations of TC, FC, and CE were determined using the TC/FC Quantification Assay (n=3 per group). The data are represented as the means ± SD of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group.