Figure 5

GSK-3β signaling pathways are involved in iron nanoparticle-induced endothelial cell permeability. A, Iron nanoparticle treatment induces the inhibition of GSK-3β and the activation of Akt. HMVECs were grown to a confluent monolayer and serum-starved overnight. The cells were exposed to 50 μg/ml iron nanoparticles for different periods of time as indicated. After the stimulation, the cells were lysed. The lysates were resolved on SDS-PAGE gel, followed by probing with different antibodies as indicated. B, PI3K is involved in iron nanoparticle-induced Akt activation and GSK-3β inhibition. HMVECS were pretreated with 10 μM LY294002, a PI3K inhibitor, for one hour, and then exposed to 50 μg/ml iron nanoparticles for 30 minutes, followed by Western blot analysis. C, ROS production is involved in iron nanoparticle-induced Akt activation and GSK-3β inhibition. HMVECs were pretreated with either 200 μM hydrogen peroxide (H₂O₂) or 10,000 units/ml catalase for one hour, and then exposed to 50 μg/ml iron nanoparticles for 30 min, followed by Western blot analysis. D, The inhibition of GSK-3β enhances iron nanoparticle-induced microtubule remodeling and cell permeability in HMVECs. HMVECs were pretreated with 5 μM GSK-3β inhibitor I for one hour, and then exposed to 50 μg/ml iron nanoparticles for 30 minutes, followed by Western blot analysis (1) and confocal microscopy analysis (2). Blue color stains for actin filaments, red color stains for acetylated α-tubulin, and green color stains for VE-cadherin. The size of the scale bar is 20 μm.