Supplementary Material for: Interleukin-24 Suppresses the Growth of Vascular Smooth Muscle Cells by Inhibiting H₂O₂-Induced Reactive Oxygen Species Production

Background/Aim: 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. Methods: 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 H₂O₂-treated mouse vascular aortic smooth muscle (MOVAS) cells. To elucidate the effect of IL-24 on ROS-induced signaling, Western blot analysis was employed. Results: IL-24 inhibited the growth of normal MOVAS cells treated with H₂O₂ by inducing a cell cycle arrest at the G₀/G₁ 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 H₂O₂. Interestingly, IL-24 attenuated the H₂O₂-induced ROS production by reducing the mitochondrial H₂O₂ production and enhancing the expression of antioxidant enzymes. We also showed that the ability of H₂O₂ to induce the PI3K/Akt and Erk signaling pathways was blocked by IL-24. Conclusion: These findings suggest a novel mechanism in which IL-24 suppresses the growth of normal VSMCs by inhibiting H₂O₂-induced ROS production through the regulation of mitochondrial ROS production and expression of antioxidant enzymes.