Fig 1: Interleukin-18 (IL-18) upregulates TRAF3IP2 but suppresses RECK expression in primary human aortic smooth muscle cells (ASMC). (A–C) IL-18 upregulates TRAF3IP2 expression. ASMCs were grown in complete media, and at 70–80% confluency, made quiescent for 48 h, and then incubated with rhIL-18 (10 ng/mL) for up to 12 h (experimental design in (A)). TRAF3IP2 mRNA expression was analyzed by RT-qPCR using a TaqMan™ probe (B) and its protein levels by Western blotting (C), with GAPDH and Tubulin serving as loading controls, respectively. * p < 0.05, ** at least p < 0.01 vs. untreated controls (n = 4). (D,E) Quiescent ASMCs were incubated with IL-18 as in (A) and were analyzed for RECK mRNA expression by RT-qPCR using a TaqMan™ probe (D) and protein levels by Western blotting (E). * p < 0.05, ** at least p < 0.01 vs. untreated controls (n = 4). (F–H) Specificity of IL-18 on TRAF3IP2 induction (G) and RECK suppression (H) was verified by incubating with neutralizing IL-18R1 antibody or IL-18BP-Fc chimera for 1 h prior to IL-18 addition for 2 (G) or 6 h (H), with normal goat IgG or Fc serving as controls. mRNA expressions of TRAF3IP2 and RECK were analyzed by RT-qPCR. (C,E) While a representative immunoblot is shown, the intensities of immunoreactive bands from 4 independent experiments were semiquantified by densitometry and are summarized on the right. (G) * p < 0.05, ** p < 0.01 vs. untreated controls, † p < 0.01 versus IL-18 (n = 6); (H) * p < 0.05, ** p < 0.01 vs. untreated controls, † p < 0.01 versus IL-18 (n = 4).