Cell culture medium EBM-2 was obtained from Lonza (Boston, MA). Fetal bovine serum was obtained from Atlanta Biologicals (Lawrenceville, GA). Fe2O3 nanoparticles were purchased from nGIMAT (Atlanta, GA). Acetylated-tubulin antibody, total tubulin antibody, actin antibody, catalase, hydrogen peroxide, EGF growth supplement, and hydrocortisone were from Sigma (St. Louis, MO). Protease and phosphatase inhibitor cocktail was from Pierce (Rockford, IL). LY294002, GSK-3β inhibitor I, nocodazole and paclitaxel were obtained from Calbiochem (La Jolla, CA). Penicillin and Streptomycin antibiotics, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), secondary antibodies-conjugated with FITC, TRITC, and Cy5 were purchased from Invitrogen (Eugene, OR). Phospho-Akt (ser-473) and total Akt antibodies were from Cell Signaling Technology (Boston, MA). Phospho-GSK-3β (ser-9) and total GSK-3β antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). VE-Cadherin antibody was purchased from Alexis (San Diego, CA).
The human microvascular endothelial cells (HMVECs) were a kind gift from Dr. Rong Shao (Biomedical Research Institute, Baystate Medical Center/University of Massachusetts at Amherst, Springfield, MA, USA). The cells were cultured according to the protocol described previously . Briefly, HMVEC were grown in endothelial basal medium-2 (EBM-2) (Lonza, Boston MA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 100 U/ml penicillin and 10 μg/ml streptomycin, 1 μg/ml of epidermal growth factor and 50 μg/ml hydrocortisone. The cells were maintained in an incubator at 37°C with 5% CO2.
Particles preparation and size measurements
Fe2O3 nanoparticles were purchased from nGIMAT (Atlanta, GA). Their surface area was approximately 165 m2/g, the average powered particle size was <10 nm, and there were trace amounts of Lead and Bismuth potentially in the particles. The iron nanoparticles were suspended in 0.1% fetal bovine serum (FBS) cell culture media at a concentration of 2.5 mg/ml. Once the nanoparticles were dispersed in 0.1% FBS cell culture media, the suspension was indirectly sonicated at 4°C for 10 min with a Hielscher-Ultrasound Technology Sonicator (UIS 259L) at amplitude 100% and cycle 1. After the indirect sonication, the suspension was further directly sonicated at 4°C for 5 min at a duty cycle setting of 10% and output of 5 with a Branson 450 Sonifier probe sonicator. The stock solution (2.5 mg/ml) of iron nanoparticles was kept at 4°C and used within 2 weeks for the experiments. The working concentration of iron nanoparticle was 50 μg/ml. Prior to being diluted to the working concentration, the stock solution was directly sonicated at 4°C for 1 min at the setting indicated above. The particle size was determined by both dynamic light scattering using Nanotrac 252 (Microtrac, Montgomeryville, PA)  and by a transmission electron microscope.
Lactate dehydrogenase (LDH) assay
The LDH release assays were measured using the LDH assay kit form Roche Diagnostics Inc. (Indianapolis, IN) according to the manufacturer instructions.
ROS production in Cell-free system
The measurements of ROS production in cell-free system were performed according to the previously published methods. Briefly, 7.5 μl of 10 mM CM-H2DCF-DA was chemically hydrolyzed to CM-H2DCF in 1 ml of 0.01 N NaOH in dark for 30 min at the room temperature, followed by neutralizing with 0.5 ml of 0.1 M PBS (pH 7.4). The reaction mixture was freshly prepared by mixing 400 μl of the neutralized CM-H2DCF, 1.6 ml of EBM-2 medium (0.1% FBS), and 0.2 units of horse radish peroxidase (HRP) to obtain a final concentration of 10 μM CM-H2DCF. Then, 2 μl of iron nanoparticle stock solution (25 mg/ml) or 2 μl of H2O2 stock solution (0.5 M) was added into 2 ml of the reaction mixture to make a final concentration of 50 μg/ml iron nanoparticles or 500 μM H2O2, respectively. After 30 minute incubation, fluorescence generated from the oxidation of CM-H2DCF to DCF was measured using a cytoflour series 4000 plate reader (PerSeptive Biosystems, Inc., Framingham, MA) at 485 nm excitation and 530 emission
ROS measurements by flow cytometry
ROS measurements by flow cytometry analysis were performed according to the methods described previously . HMVECs were pretreated with 10 μM CM-H2DCFDA for 60 min. After the pretreatment, the cell culture media was removed and replaced with the media containing iron nanoparticles (50 μg/ml) and 10 μM CM-H2DCFDA for further stimulation. After the stimulation, the cells were quenched on ice for 10 min then washed three times with ice-cold PBS before they were harvested by scrapping. The cells were fixed with 10% formaldehyde for 20 min at room temperature and then washed three times with PBS, followed by resuspension in 400 ml of PBS. ROS measurements were carried out by a flow cytometry using FACSCalibur system (BD Biosciences, Rutherford, NJ) with a 488-nm excitation beam. The signals were obtained using a 530-nm band-pass filter for CM-H2DCFDA. Each measurement was based on the mean fluorescence intensity of 10,000 cells.
Transendothelial electrical resistance
The transendothelial electrical resistance (TER) was measured using electrical cell-substrate impedance sensing system (ECIS) (Applied Biophysics, Troy, NY) according to the published protocol . Briefly, HMVECs were grown to confluent monolayer on ECIS culture ware and serum-starved overnight. The electrical resistance was measured on cells located on the small gold electrodes in each of the wells. The culture medium was the electrolyte. The small gold electrode covered by confluent HMVECs and a larger gold counter electrode were connected to a phase-sensitive lock-in amplifier. A constant current of 1 μA was supplied by a 1-V, 4,000-Hz alternating current through a 1-MΩ resistor. Changes in voltage between the small electrode and the large counter electrode were continuously monitored by the lock-in amplifier, stored, and then calculated as resistance.
Immunofluorescence assay and Western blot analysis
Immunofluorescence assays were performed according to the methods published previously . Briefly, HMVECs were grown on coverslides. After treatment, cells were fixed and permeabilized, followed by labeling with the specific antibodies for the targeted proteins as well as immunofluorescence-conjugated secondary antibodies. The labeled coverslides were mounted to the slides with antifade reagent (Invitrogen, Eugene, OR). A Zeiss LSM 510 microscope was used to obtain images. Scale bars were generated and inserted by LSM software. Western blot analysis was performed according to the methods described previously . Briefly, the cell lysates were resolved in 8% SDS-PAGE gel, and then transferred to PVDF membranes, followed by blotting with different antibodies for the individual targeted proteins. Horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) were applied to visualize proteins using chemiluminescence.
Transmission electron microscopy (TEM) of iron nanoparticles
TEM of iron nanoparticles was performed according to previously published procedures . Briefly, HMVECs were grown and stimulated in transwell tissue polycarbonate membrane polystyrene plates, and were then washed with ice cold PBS. The cells were fixed in Karnovsky's fixative (2.5% glutaraldehyde + 3% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4), and then washed three times in 0.1 M sodium cacodylate and post-fixed in 1% osmium tetra oxide, followed by washing with 0.1 M sodium cacodylate and distilled water. The cells were dehydrated by sequential washings in 25%, 50% and 100% ethanol then embedded in LX-112 (Ladd, Williston, VT). The ultrathin sections were stained with uranyl acetate and lead citrate and examined with a TEM (JEOL 1220, Tokyo, Japan). To measure the size distribution of iron nanoparticles in the cell culture medium, iron nanoparticles (50 μg/ml) were prepared as indicated above. An aliquot of this working solution was then dropped on a formvar-coated grid, let to dry then analyzed by transmission electron microscopy.
Dynamic light scattering measurements
Suspension of iron nanoparticles at 50 μg/ml was prepared in 0.1% FBS EBM-2 media. The iron nanoparticle suspension was sonicated with a probe sonicator (Branson Sonifier 450, 10 W continuous output) for 30 min and then vortexed for 1 min, followed by measuring the particle size by dynamic light scattering using Nanotrac 252 (Microtrac, Montgomeryville, PA). During sonication, heat was dissipated by placing the samples on ice .