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Fig. 5 | Molecular Medicine

Fig. 5

From: Inhibition of HMGB1/RAGE-mediated endocytosis by HMGB1 antagonist box A, anti-HMGB1 antibodies, and cholinergic agonists suppresses inflammation

Fig. 5

Cholinergic agonists acetylcholine and GTS-21 inhibited HMGB1 endocytosis, reduced HMGB1 and LPS-induced TNF release in macrophages. a. Primary human macrophages (upper) or thioglycollate-elicited mouse macrophages (lower) seeded on 24-well culture plate on cover slips (1 × 104/well) were incubated with Alexa 555- HMGB1 (1 μg/ml) alone or plus GTS-21 or acetylcholine for 2 h at 37 °C. Endocytosis results of Alexa 555-HMGB1 were expressed as fold of Alexa 555-HMGB1 alone. N = 32–48 cells per treatment. *: P < 0.05 vs. Alexa 555-HMGB1 alone. b. RAW 264.7 cells in 96-well culture plates were used at 90% confluence. Cells were stimulated with HMGB1 or LPS, with the addition of GTS-21, acetylcholine (Ach) as indicated for 16 h. Pyridostigmine bromide (PB) was added (1 μM). TNF released was measured using ELISA. N = 3 experiments. *: P < 0.05 vs. HMGB1 or LPS alone. c. RAW 264.7 cells in 96-well culture plates (90% confluence) were incubated with Ach, GTS-21, K883, box A, m2G7, mouse IgG (mIgG), or Dynasore (DYN) as indicated for 16 h. Cell lysate (positive control) or supernatants were collected and assayed for LDH content (AU). AU: arbitrary unit. N = 3

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