were developed with the Luminescent Image Analyzer LAS-3000 (Fujifilm). Aortic sprouting Isolation and preparation of aortic rings were performed essentially as described [25]. In brief, aortas were isolated from 8–12 – week old C57BL/6 mice, transferred into 5 ml Opti-MEM (GIBCO), carefully cleaned of the protective fatty layer and all accessory vessels, and sectioned into 0.5 mm rings. The rings were incubated at 37uC for 2 hours in medium, embedded into growth factor reduced Matrigel (BD), and treated with HKa (100 nM) in presence or absence of spleen ferritin (200 nM) in Opti-MEM supplemented with 2.5% FBS and Penicillin/Streptomycin (GIBCO) plus 30 ng/ml recombinant mouse VEGF (R&D Systems) for 48 hours. Media was replaced and subsequently changed every two days. Aortic rings were photographed at day 5 post treatment. Quantification was performed by counting the number of sprouts originating from the aortic rings. Cell adhesion assay Six-well cell culture plates were coated with 2 ng/ml vitronectin (Promega), blocked with 0.1% bovine serum albumin (BSA) and washed with phosphate buffered saline (PBS). HUVECs (46105cells/well) were plated in medium containing 20 ng/ml bFGF and 10 mM of ZnCl2 and treated with either HKa or HGK- rich peptide in the presence or absence of ferritin. When control cells were adherent (1 to 2 hours at 37uC), all samples were lysed in TX-100 lysis buffer and subjected to Western blotting. To count adherent cells, coverslips were washed with PBS, fixed in 10:10:80 acetic acid:methanol:water, and treated with 0.4% crystal violet. Expression and purification of recombinant HFt and LFt cDNA encoding the human ferritin H subunit was cloned into the pET-17 bacterial expression vector (Novagen) and trans- formed into E.coli BL21(DE3) for expression. Following overnight induction with 1 mM IPTG, cell pellets were collected and resuspended in 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM PMSF, and 10% sucrose. Cells were lysed using an Emulsiflex C-5 cell homogenizer, and cell debris pelleted. The supernatant was heated at 70uC for 10 min, clarified by centrifugation, and passed over a Q-sepharose (Amersham) column equilibrated with 50 mM Tris-HCl pH 7.5. The column was eluted with a gradient of 20 mM-2 M NaCl and the fractions containing HFt were pooled and loaded onto a Superdex S-200 (GE Healthcare) gel filtration column equilibrated with 50 mM Tris-HCl pH 7.5, 200 mM NaCl. Fractions containing HFt were pooled, analyzed for purity via SDS-PAGE electrophoresis, and concentrated to 10 mg/mL. LFt cDNA was similarly cloned into the pET-17 vector and expressed and purified using the same protocol. Endotoxin was removed using Detoxi-gel endotoxin removing gel (Thermo Scientific) and quantified using QCL-1000 Quantitative Chromo- genic LAL Assay (Lonza). Expression and purification of recombinant HK5 cDNA encoding domain 5 of the human high molecular weight kininogen protein (HK5) was subcloned into the pGEX-6P-1 bacterial expression vector (GE Healthcare) in frame with glutathione S-transferase (GST) and recombinant protein was expressed and purified as described [20]. Biotinylation of ferritin H and ferritin L Biotinylation of recombinant HFt and LFt was performed using EZ-Link NHS-PEO4-Biotinylation Kit (Pierce) according to the Ferritin and HKa-Mediated Signaling manufacturer’s recommendations. The HABA/Avidin assay (Biotin Quantification Kit, Pierce) was used to estimate molar biotin incorporation. GST-pull-down of recombinant proteins Protein G Sepharose beads (GE Healthcare Biosciences) were washed and incubated with either GST-D5 plus biotinylated HFt, GST-D5 plus biotinylated LFt, GST-D5 plus the combination of biotinylated HFt and LFt, or GST-D5 plus non-biotinylated HFt or LFt at 4uC overnight. Anti-GST antibody (Sigma) was added and incubation continued for an additional 2 hr at 4uC. Beads were washed, centrifuged, and bound complexes analyzed by western blotting. Alexa Fluor 488 One mg of HKa or D5 was labeled with Alexa Fluor 488 according to the manufacturer’s protocol (Molecular Probes/ Invitrogen). Samples were incubated for 1 hour at room temper- ature and labeled proteins were purified using the purification resin provided with the kit. Flow cytometric analysis Five 6105 HUVEC cells were treated with 50 nM Alexa-Fluor 488-labeled HKa in the presence or absence of 100 nM unlabeled ferritin, 100 nM BSA, or 50-fold excess unlabeled HKa in basal medium containing 20 ng/ml bFGF and 10 mM ZnCl2 for one hour at 37uC. Cells were treated in suspension, washed with 1% FBS in PBS and analyzed on FACSCaliber using CellQuest Pro Software. Co-immuno-precipitation of UPAR and Integrins a5b1 Endothelial cells were plated on 2 ng/ml vitronectin coated plates. When cells were adherent, the culture medium was replaced with M199 basal medium overnight. Cells were either left untreated or treated with HKa in the presence or absence of ferritin in medium containing 20 ng/ml of bFGF and 10 mM ZnCl2 for 24 hours. Cells were lysed in TX-100 lysis buffer and protein concentration of clarified samples determined using BCA protein assay kit (Pierce). For immuno-precipitation 500 mg of cell lysates were incubated with an anti-a5b1 antibody (Millipore) at 4uC overnight with agitation. Beads were washed with 1% BSA in lysis buffer, added to samples, and incubated for 2 hours. Bound complexes were centrifuged and subjected to SDS-PAGE. Membranes were probed with anti-UPAR antibody (Santa Cruz) and anti-b1 integrin antibody (Millipore). Ligand blot Ferritin binding to immobilized recombinant proteins was determined by a ligand blot as previously described [21]. Statistical Analysis Results were analyzed by one-way ANOVA followed by pairwise comparison using Students’ t-test. Data was considered significant at p#0.05. Supporting Information Figure S1 Effects of ferritin on HKa-Mediated inhibi- tion of endothelial cell viability are dose-dependent. HUVECs were treated with nothing (CTL) or 50 nM HKa in the presence of increasing concentrations of Ft (0–400 nM) for 24 hours and viability assessed using an MTT assay. Cells were PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40030