Fig. 6.
Macrophage endocytosis of LPS-HMGB1 complexes was inhibited by m2G7, box A and acetylcholine. a. Upper: RAW 264.7 cells, seeded on 24-well culture plate on cover slips (1 × 104/well), were incubated with Alexa 555 labeled HMGB1 (1 μg/ml) alone or in combination with increasing amounts of LPS as indicated for 2 h at 37 °C. Endocytic results were expressed as fold change of Alexa 555-HMGB1 alone. N = 30–35 cells per treatment and are representative from 6 independent experiments. Middle and lower: RAW 264.7 cells, seeded on 24-well culture plate on cover slips (1 × 104/well), were incubated with Alexa 568 labeled LPS (# red, 1 μg/ml) in complex with HMGB1 as indicated. N = 28–30 cells for each treatment. Endocytic uptake was expressed as fold change of Alexa 555-HMGB1 (upper) or Alexa 568-LPS (lower) alone. Scale bar = 10 μm. *: P < 0.05 vs. Alexa 555-HMGB1 or Alexa 568-LPS alone. b. RAW 264.7 cells, on 24-well culture plate on cover slips (1 × 104/well), were incubated with Alexa 555-HMGB1 (1 μg/ml) in complex with LPS (1 ng/ml), plus m2G7, mouse IgG or box A as indicated for 2 h at 37 °C. Endocytic uptake of Alexa 555-HMGB1 was expressed as fold change of Alexa 555-HMGB1 + LPS. *: P < 0.05 vs. Alexa 555-HMGB1 + LPS. N = 30–40 cells for each treatment. c. RAW 264.7 cells, on 24-well culture plate with cover slips (1 × 104/well), were incubated with Alexa 568 labeled LPS (# red, 1 μg/ml) in complex with HMGB1 (1 μg/ml), together with m2G7, mouse IgG or box A as indicated for 2 h at 37 °C. Endocytic uptake of Alexa 568 LPS was expressed as fold of Alexa 568-LPS + HMGB1. Scale bar = 10 μm. *: P < 0.05 vs. Alexa 568-LPS + HMGB1. N = 30–35 cells for each treatment. d. RAW 264.7 cells, seeded on 24-well culture plate on cover slips (1 × 104/well), were incubated with Alexa 568 labeled LPS (# red, 1 μg/ml) in complex with HMGB1 (1 μg/ml), together with acetylcholine as indicated for 2 h at 37 °C. Endocytic uptake of Alexa 568 LPS was visualized, analyzed and quantified using Image J. *: P < 0.05 vs. Alexa 568-LPS + HMGB1. N = 30–35 cells for each treatment. Scale bar = 10 μm