Research Paper Volume 13, Issue 18 pp 22412—22431

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

Pim-2 suppresses atherosclerotic inflammation via the mTORC1 pathway in THP-1-derived macrophages. (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 in Si-mTOR- or Si-Raptor-treated ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. 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 Si-mTOR- or Si-Raptor-disposed ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. (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 Si-mTOR- or Si-Raptor-treated ox-LDL-treated THP-1-derived macrophages after Pim-2 KD; the values were 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). Data are represented as the means ± SD of three independent experiments; *P P P #P

Figure 8. Pim-2 suppresses atherosclerotic inflammation via the mTORC1 pathway in THP-1-derived macrophages. (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 in Si-mTOR- or Si-Raptor-treated ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. 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 Si-mTOR- or Si-Raptor-disposed ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. (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 Si-mTOR- or Si-Raptor-treated ox-LDL-treated THP-1-derived macrophages after Pim-2 KD; the values were 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). Data are represented as the means ± SD of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 versus the NC group; #P < 0.05 versus the ox-LDL-treated NC group.