Supplementary Material for: Interleukin-24 Suppresses the Growth of Vascular Smooth Muscle Cells by Inhibiting H<sub>2</sub>O<sub>2</sub>-Induced Reactive Oxygen Species Production K.-M.Lee H.-A.Kang M.Park H.-Y.Lee M.-J.Song K.Ko J.-W.Oh H.-S.Kang 2012 <b><i>Background/Aim:</i></b> The abnormal growth of vascular smooth muscle cells (VSMCs) induced by reactive oxygen species (ROS) is considered a major pathogenic process in vascular diseases. Interleukin (IL)-24 specifically inhibits cancer cell growth through the induction of cell cycle arrest and apoptosis. However, the role of IL-24 in ROS-induced VSMC growth has not yet been investigated. <b><i>Methods:</i></b> An MTT assay, gene expression analysis, flow cytometry and a scratch wound healing assay were performed to determine the anti-growth effects of IL-24 in H<sub>2</sub>O<sub>2</sub>-treated mouse vascular aortic smooth muscle (MOVAS) cells. To elucidate the effect of IL-24 on ROS-induced signaling, Western blot analysis was employed. <b><i>Results:</i></b> IL-24 inhibited the growth of normal MOVAS cells treated with H<sub>2</sub>O<sub>2</sub> by inducing a cell cycle arrest at the G₀/G<sub>1</sub> phase through the regulation of p21 and cyclin D1. Furthermore, IL-24 suppressed mRNA expression of vascular endothelial growth factor and platelet-derived growth factor and subsequently decreased the level of cell migration in response to H<sub>2</sub>O<sub>2</sub>. Interestingly, IL-24 attenuated the H<sub>2</sub>O<sub>2</sub>-induced ROS production by reducing the mitochondrial H<sub>2</sub>O<sub>2</sub> production and enhancing the expression of antioxidant enzymes. We also showed that the ability of H<sub>2</sub>O<sub>2</sub> to induce the PI3K/Akt and Erk signaling pathways was blocked by IL-24. <b><i>Conclusion:</i></b> These findings suggest a novel mechanism in which IL-24 suppresses the growth of normal VSMCs by inhibiting H<sub>2</sub>O<sub>2</sub>-induced ROS production through the regulation of mitochondrial ROS production and expression of antioxidant enzymes.