Fig. 3

AMPK activation promotes engulfment of HMGB1 and facilitates degradation in lysosome in mouse BMDMs. A rHMGB1 (his tag) contents in BMDMs treated with AMPK activators AICAR (300 μM), A-769662 (100 μM), and metformin (2.5 mM) (n = 3). B rHMGB1 (his tag) contents in BMDMs treated with metformin (2.5 mM) in the presence of protease inhibitor (MG132, 10 μM) or lysosomal inhibitor (CQ, 20 μM) (n = 3). C rHMGB1 (his tag) contents in BMDMs treated with AICAR (300 μM) or A-769662 (100 μM) in the presence of CQ (20 μM) (n = 3). BMDMs were administered with inhibitor MG132 or CQ for 15 min, then treated with AMPK agonist (AICAR or metformin) for 15 min, followed by co-culturing with rHMGB1 (his tag) (10 nM) for 1 h, and subsequently collected for western blotting. D Confocal microscopy images of HMGB1-FITC (green) and Lysotracker (red) co-localization in BMDMs. Cells were pretreated with metformin (2.5 mM) for 15 min and then co-cultured with HMGB1-FITC (10 nM) and Lysotracker (75 nM) for 1 h. Scale bar: 20 μm. Representative bands and a data summary (n = 3 for each group) are shown. A significant difference was revealed following one-way ANOVA (*p < 0.05, **p < 0.01 and ***p < 0.001 vs. Control; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. AICAR group or metformin group; Bonferroni post hoc tests)