Figure 4

PARylated HMGB1 binds with increased affinity to PS on apoptotic cells and to RAGE on macrophages compared with unmodified HMGB1. (A) Apoptotic thymocytes or WT macrophages were incubated with 1 μg/mL HMGB1 or PARylated HMGB1 for 2 h at 37°C or left untreated (Control), and then HMGB1 binding was examined by immunofluorescence. Representative images are shown. Two additional independent experiments provided similar results. (B) Quantification of HMGB1 binding to apoptotic cells or macrophages using ImageJ software (n = 10 cells from three independent experiments). Means ± SEM are shown. *P < 0.05 and **P < 0.01 compared with control. (C, D) PARylated HMGB1 does not bind to viable thymocytes and binds with diminished affinity to RAGE^-/- macrophages. Viable thymocytes (C) or RAGE^-/- macrophages (D) were incubated with 1 μg/mL PARylated HMGB1 for 2 h at 37°C, and then HMGB1 binding was examined by immunofluorescence. Representative images are shown. Two additional independent experiments provided similar results.