HMGB1- and H4-mediated expression of CAMs in HUVECs in the absence and presence of polyP-70. (A) Confluent endothelial cells were incubated with increasing concentrations of HMGB1 (10-40 nM for 16 hours) followed by measuring the cell surface expression of ICAM-1 (white bar), VCAM-1 (gray bar), and E-selectin (black bar) by a cell-based enzyme-linked immunosorbent assay (ELISA). (B) The same as (A), except that H4 (0.22-3.5 µM) was used for endothelial cell activation. (C) The same as (A), except that polyP-mediated amplification of signaling by low concentrations of HMGB1 (5-10 nM) was monitored. (D) The same as (A), except that polyP-mediated amplification of signaling by low concentrations of H4 (0.22-0.44 µM) was monitored. (E) The same as (A), except that the competitive effect of sRAGE on CAM expression by HMGB1 (40 nM) was monitored. (F) The same as (E), except that the competitive effect of sRAGE on the polyP-mediated amplification of signaling by a low concentration of HMGB1 (10 nM) was monitored. (G) The same as (E), except that the competitive effect of sRAGE on the polyP-mediated amplification of signaling by a low concentration of H4 (0.44 µM) was monitored. (H) Analysis of caspase-8 activity by a colorimetric assay. (I) Analysis of NF-κB activation by different stimuli (lane 1, buffer control; lane 2, 40 nM HMGB1; lane 3, 3.5 µM H4; lane 4, 50 µM polyP; lane 5, 2.5 µM polyP-70 + 10 nM HMGB1; lane 6, 2.5 µM polyP-70 + 0.44 µM H4). (J) Densitometric analysis of NF-κB data in (I). All results are shown as mean ± standard deviation of 3 different experiments. *P < .05; **P < .01; ***P < .001.