Figure 9

Macrophage migration inhibitory factor restores cell rejuvenation through CD74-dependent AMPK–FOXO3a signaling. (A) Proliferation growth curves of mesenchymal stem cells (MSCs) transfected with small interfering RNA (siRNA)-AMP-activated protein kinase (AMPK), siRNA-Forkhead box class O 3a (FOXO3a), or scrambled small interfering RNA (siRNA-NT) control, and treated with macrophage migration inhibitory factor (MIF). Each data point represents mean ± standard deviation from three independent experiments; *P <0.05 versus control; ^△ P <0.05 versus MIF + siRNA-AMPK; ^◇ P <0.05 versus MIF + siRNA-FOXO3a. Concentration of (B) vascular endothelial growth factor (VEGF), (C) basic fibroblast growth factor (bFGF), (D) hepatocyte growth factor (HGF) and (E) insulin-like growth factor (IGF) under normal and hypoxic conditions, in the culture medium of MSCs transfected with siRNA-AMPK, siRNA-FOXO3a or siRNA-NT control, and treated with MIF. Each column represents mean ± standard deviation from three independent experiments; *P <0.05 versus control; ^△ P <0.05 versus MIF + siRNA-AMPK; ^◇ P <0.05 versus MIF + siRNA-FOXO3a. (F) Representative distributions of propidium iodide (PI) and Annexin V staining from FACScan flow cytometric analyses of apoptotic cells in normal and hypoxia and serum deprivation (hypoxia/SD) (6 hours) conditions, in cultures of MSCs transfected with siRNA-AMPK, siRNA-FOXO3a or siRNA-NT control, and treated with MIF (100 ng/ml at the point of exposure to hypoxia/SD). MIF was added to the incubation medium throughout the hypoxia/SD treatment period. (G) Fold-change of apoptotic cells in the above conditions, compared with control. Each column represents mean ± standard deviation from three independent experiments; *P <0.05 versus control; ^△ P <0.05 versus hypoxia/SD + MIF; ^◇ P <0.05 versus hypoxia/SD + siRNA-NT.