Fig. 3.

Anti-HMGB1 m2G7 and box A inhibited macrophages endocytosis of HMGB1. a. Murine macrophage-like RAW 264.7 cells seeded on 24-well culture plate on cover slips (1 × 10⁴/well) were incubated with Alexa 555 labeled (red) isoforms of HMGB1 (disulfide, sulfonyl and fully reduced) for 2 h at 37 °C. Endocytic uptake of HMGB1 was visualized via fluorescence microscope. Nuclei were counterstained with DAPI (blue). Right panel shows corresponding phase contrast image of cells. Data are representative from 4 independent experiments. Scale bar = 10 μm. b. RAW 264.7 cells seeded on 24-well culture plate on cover slips (1 × 10⁴/well) were incubated with Alexa 555 labeled HMGB1 (^# red) alone or plus Dynasore (DYN, 8 μM), m2G7 (5 μg/ml), or box A (50 μg/ml) for 2 h at 37 °C. Endocytic uptake of HMGB1 was visualized. Right panel shows corresponding phase contrast image of cells. Data are representative from 3 to 4 independent experiments. Scale bar = 10 μm. c. Thioglycollate-elicited mouse primary macrophages (upper) or RAW 264.7 cells (lower) were seeded on 24-well culture plate on cover slips (1 × 10⁴/well) and incubated with Alexa 555-HMGB1, alone or with box A for 2 h at 37 °C. HMGB1 endocytosis results expressed as fold of Alexa 555-HMGB1 alone. N = 30–42 cells per treatment. *: P < 0.05 vs. HMGB1 alone. d. Thioglycollate-elicited mouse primary macrophages (upper) or RAW 264.7 cells (lower) were seeded on 24-well culture plate on cover slips (1 × 10⁴/well) and incubated with Alexa 555 labeled HMGB1 (1 μg/ml) alone or with m2G7 or mouse IgG for 2 h at 37 °C. Endocytosis results expressed as fold of Alexa 555-HMGB1 alone. N = 32–44 cells per treatment. *: P < 0.05 vs. HMGB1 alone