Fig 5.

GSK2593074A (GSK’074) directly inhibits vascular smooth muscle cell (VSCM) necroptosis and macrophage migration. A, Mouse primary smooth muscle cells were treated with 100 ng/mL tumor necrosis factor-α (TNF-α) plus 60 μmol/L zVAD and 10 nmol/L GSK’074 for 24 hours. Cells were then stained with 7-AAD and analyzed via flow cytometry. B, Mouse aortic smooth muscle cells were treated with 30 ng/ml TNF-α plus 60 μmol/L zVAD overnight in the presence or absence of 10 nmol/L GSK’074. Receptor interacting proteins kinase (RIPK)1 and RIPK3 complex formation were detected by coimmunoprecipitation with anti-IgG or anti-RIPK3 antibodies followed by Western blotting analysis with the indicated antibodies. C, Mouse bone marrow-derived macrophages (BMDMs) were pretreated with 100nM GSK’074 or dimethylsulfoxide (DMSO) for 2 hours, cell migration toward MCP1 (100ng/ml) was determined via transwell migration assay. One-way analysis of variance with a post hoc Tukey's test was performed. D, Mouse BMDMs were pretreated with 100 nmol/L GSK’074 or DMSO for 2 hours followed by 1 ng/mL lipopolysaccharide (LPS) stimulation in the presence or absence of GSK’074 for 6 hours. Messenger RNA levels of Il1b and Tnf were determined by real-time polymerase chain reaction. One-way analysis of variance with a post hoc Tukey's test was performed. Data are presented as mean ± standard error of three independent experiments. ∗P ≤ .05. ns, Nonsignificant.