Fig. 5

HMGB1 binds to RAGE on TAMs. A Co-IP assays of interaction of HMGB1 with RAGE in THP1 cell line-derived macrophages (left panel) and RAW264.7 macrophages (right panel). Cell lysates were immunoprecipitated with anti-HMGB1 antibody, then immunoblotted with anti-HMGB1 and anti-RAGE antibodies. B Co-IP assays of interaction of RAGE with HMGB1 in THP1 cell line-derived macrophages (left panel) and RAW264.7 macrophages (right panel). Cell lysates were immunoprecipitated with anti-RAGE antibody, then immunoblotted with anti-HMGB1 and anti-RAGE antibodies. C THP1 cell line-derived macrophages (left panel) and RAW264.7 macrophages (right panel) were treated with RFP-labeled rhHMGB1 or FITC-labeled rmHMGB1 for 5 and 30 min, respectively. Scale bars = 10 μm. D Colocalization of HMGB1 (green) and RAGE (red) in human GB samples (GB8060 and GB9080) with TMZ treatment. Scale bars = 10 μm. E The mRNA expression of HMGB1 receptors (TLR2, TLR4, TLR9, RAGE) by THP1 cell line-derived macrophages after stimulation with rhHMGB1 at indicated concentrations for 24 h (left panel). Immunoblot of RAGE in THP1 cell line-derived macrophages with or without rhHMGB1 treatment for 48 h (right panel). F The mRNA expression of HMGB1 receptors (Tlr2, Tlr4, Tlr9, Rage) by RAW264.7 macrophages after stimulation with rmHMGB1 at indicated concentrations for 24 h (left panel). Immunoblot of RAGE in RAW264.7 macrophages with or without rmHMGB1 treatment for 48 h (right panel). *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance