Ferritin and HKa-Mediated Signaling Figure 3. Ferritin modulates HKa-dependent effects on proliferation and adhesion by targeting an HGK-rich region of HKa. A. HUVECs were treated with 10 nM or 50 nM HKa and 100 nM ferritin for 24 hours in basal medium containing 10 mM ZnCl2 in the absence of growth factors. Viability was assessed using an MTT assay. B. Cells were treated as in A in the presence of 20 ng/ml bFGF. C. HUVEC cells grown in basal medium containing 10 m M ZnCl2 and 20 ng/ml bFGF were treated with 300 nM D5, 2 mM HGK-rich peptide or 2 mM control peptide in the presence or absence of 100 nM ferritin for 24 hours and viability assessed using an MTT assay. Shown are means and standard deviation of triplicate experiments with * p,0.004 and ** p,0.0004. D. Cells were allowed to adhere to vitronectin coated dishes for two hours in the presence of 2.5 mM HGK peptide, 100 nM ferritin, or the combination of HGK peptide and ferritin. Phosphorylation of Erk1/2 and paxillin was assessed by western blotting. F. Phosphorylation of signaling molecules was quantified by densitometry. Means and standard deviation of 4 independent experiments are shown. *p,0.01; **p,0.002. doi:10.1371/journal.pone.0040030.g003 HKa exhibited specific binding to HUVECs that was competed by excess unlabeled HKa. Co-treatment of cells with the combination of HKa and ferritin (molar ratio of 1:2) reduced binding of HKa approximately 50%. Co-treatment of HKa with the control protein BSA did not affect binding. The binding of HKa to uPAR disrupts uPAR-mediated signaling. We tested whether the diminished binding of HKa to the endothelial cell surface in the presence of ferritin (Figure 6) was sufficient to block the ability of HKa to the uPAR signaling complex. Cells were incubated with either HKa or Ft alone, or with the combination of HKa plus ferritin. Signaling complexes PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40030