Fig. 4. HMGB1 moves to the cytoplasm following TSA treatment. (A) Exposure of mouse fibroblasts to 10 ng/ml TSA for 1 h causes a significant relocation of HMGB1–GFP to the cytoplasm; no vesicles are recognizable. The mutation of six lysines to arginines (2×KKK→RRR) prevents the cytoplasmic accumulation of HMGB1–GFP, even after TSA treatment. (B) U937.12 cells were cultured for 3 h with or without LPS or leptomycin B. Cells were then fixed and immunostained with anti-HMGB1 antibody (red). HMGB1 is exclusively nuclear in resting U937.12 cells, whereas it is predominantly vesicular in LPS-activated cells. Leptomycin blocks nuclear export, and consequently vesicular accumulation. A significant fraction of HMGB1 is contained in the cytoplasm and in vesicles in resting U937.12 cells that have been incubated 3 h at 4°C to promote passive diffusion of hypoacetylated HMGB1 to the cytoplasm and rewarmed to 37°C for 5 min to resume active transport (‘cooling and rewarming’). Likewise, a significant fraction of HMGB1 is contained in the cytoplasm and in vesicles when U937.12 cells enter M phase and free hypoacetylated HMGB1 into the cytoplasm after nuclear membrane breakdown (‘metaphase’). (C) Mouse peritoneal macrophages contain HMGB1 in the nucleus, but after stimulation with LPS, a fraction of HMGB1 is transferred to cytoplasmic vesicles. Incubation with TSA, but in the absence of LPS, also causes the transfer of HMGB1 to vesicles.