Fig. 1

Release of HMGB1 induced by TMZ in GB is dependent on the autophagic vacuoles. A Immunoblot of HMGB1, SQSTM1 and LC3B-I to LC3B-II conversion in primary GB1 cells (GB1 cells) treated with 1000 μM TMZ (left panel). ELISA determining the HMGB1 in supernatants of primary GB1 cells (GB1 cells) (right panel). n = 3. B Immunoblot of HMGB1, SQSTM1 and LC3B-I to LC3B-II conversion in GB1 cells treated with TMZ for 72 h (left panel). ELISA determining the HMGB1 in supernatants of GB1 cells (right panel). n = 3. C Colocalization of LC3B (red) with HMGB1 (green) in GB1 cells under TMZ (left panel). Colocalization tracer profile along the line (white arrows) is indicated as merged images (middle panel). Pearson’s colocalization coefficient for LC3B and HMGB1 derived from three independent experiments with five fields (right panel). Scale bars = 10 μm. D Immunoblot of HMGB1 and LC3B-I to LC3B-II conversion in GB1 cells with or without 3-MA (5 mM) and LY294002 (100 nM) for 4 h, then stimulated with or without TMZ (1000 μM) for 24 h (left panel). HMGB1 was detected in the supernatants by ELISA (right panel). E Immunoblot of ATG5 and LC3B-I to LC3B-II conversion in GB1 cells transiently transfected with scrambled siRNA (siCtrl) or siRNA ATG5 (siATG5) for 48 h then treated with TMZ (1000 μM) for 24 h (left panel). HMGB1 in the supernatants was detected by ELISA (right panel). F GB1 cells transfected with scrambled siRNA (siCtrl) or siRNA GORASP2 (siGORASP2) for 36 h were transfected with AAV-mCherryGFP-LC3B then treated with TMZ (1000 μM) for 24 h (left panel). The number of autophagosomes was analyzed in ten random fields for each independent experiment (right panel). G Immunoblot of GORASP2 and LC3B-I to LC3B-II conversion in GB1 cells transiently transfected with siCtrl and siGORASP2 for 48 h then with TMZ (1000 μM) treatment for 24 h (left panel). HMGB1 in the supernatants was detected by ELISA (right panel). *P < 0.05, **P < 0.01,***P < 0.001, ns = no significance