Figure 4

ROS production is involved in iron nanoparticle-induced cell permeability. A, Iron nanoparticles induce the production of ROS in HMVECs. 1. Iron nanoparticles induce the production of ROS in HMVECs. The monolayer HMVECs were pretreated with 10 μM CM-H₂DCFDA, as well as 10,000 unit catalase as indicated, for one hour, followed by exposure to 50 μg/ml iron nanoparticles or 500 μM H₂O₂ for another hour as indicated. After the stimulation, the cells were collected and analyzed by a flow cytometry. Values given are means ± SD (t-test, n = 5, *p < 0.01). 2. Iron nanoparticles do not induce the production of ROS in cell-free system. The hydrolyzed CM-H₂DCF-DA was mixed with 50 μg/ml iron nanoparticles, 100 μg/ml iron nanoparticles, or 1 mM H₂O₂ as indicated, followed by the fluorescence measurements using a cytoflour series 4000 plate reader. Values given are means ± SD (t-test, n = 5, *p < 0.05). B, Catalase pretreatment inhibits iron nanoparticle-induced microtubule remodeling and permeability in HMVECs. HMVECs were pretreated with 10,000 units/ml of catalase for one hour, followed by exposure to 50 μg/ml iron nanoparticles for 30 minutes. The cells were fixed and stained for acetylated α-tubulin (red color) and VE-cadherin (green color). A Zeiss confocal microscope was applied to take the images. The size of the scale bar is 20 μm. 1. Images of microtubule remodeling and cell permeability. The right panels are an overlay of two different stains. 2. Images of microtubule remodeling for the individual cells. C, Catalase pretreatment inhibits iron nanoparticle-induced microtubule stabilization. HMVECs were pretreated with 10,000 units/ml catalase for one hour, followed by the exposure to 50 μg/ml iron nanoparticles for one hour. The cells were lysed, and the lysates were resolved with 8% SDS-PAGE gel. An anti-acetylated α-tubulin antibody was applied to detect the expression of acetylated α-tubulin.