Fig. 4

OPN binds and neutralizes the cytotoxic effects of histones. a Surface plasmon resonance (SPR) sensorgrams illustrating interactions between different histone subunits (i.e., H1, H2A, H2B, H3.1, and H4) (ligand) and OPN (analyte). Histone subunits were covalently bound to a CM5 sensor chip using a standard amine-coupling kit. Different concentrations of OPN (62.5, 125, 250, 500, and 1000 nM) in running buffer were injected over the sensor chip to determine binding. The curves obtained after OPN injection were analyzed to show binding incidence with association and dissociation curves between H1, H2A, H2B, H3.1, H4, and OPN. b Possible interference of OPN with the histone-induced cytotoxic effects against human bronchial epithelial cells (BEAS-2B cells). The histones H1, H2A, H2B, H3.1, and H4 alone or a mixture of histones derived from calf thymus (CTH), or histones preincubated with OPN at a ratio of 1:1 were used. The cytotoxicity of histones is presented as percentage LDH release of the total LDH content. c Membrane permeability effects of histone subunits or CTH in the presence and absence of OPN against human endothelial cells (EA.hy926) are presented as fluorescence dye leakage. d Hemolytic activity of histone subunits or CTH in the presence and absence of OPN against human erythrocytes is presented as percentage hemolysis. OPN had a strong protective effect against both histone-induced cytotoxicity and hemolysis. The results are mean ± SEM (n = 3)