HMGB1 in vesicles is from the nucleus. (A) LMB inhibits HMGB1 translocation into TLR9-containing vesicles. BMDMs were incubated in the presence or absence of 20 ng/mL LMB for 45 minutes, followed by treatment with CpG-ODN-Cy5 (1018, 5 μg/mL). △ indicate HMGB1-containing vesicles; ▴, CpG-DNA-containing vesicles; and ↑, early CpG-DNA-containing vesicle prior to acquisition by HMGB1/TLR9-containing vesicles. (B) Quantitative analysis of the fluorescence of HMGB1 and TLR9 within vesicles over background fluorescence in BMDMs treated with or without 20 ng/mL LMB for 45 minutes (means ± SEM, n = 35, **P < .001, Student t test). (C) LMB impairs the formation of the TLR9-HMGB1 complex. HMGB1 was immunoprecipitated from lysates of WEHI-231 cells that were treated with 20 ng/mL LMB for 45 minutes prior to stimulation with CpG-ODN (10 μg/mL) for 30 minutes. (D) Exogenously added rHMGB1 can restore the presence of HMGB1 in the vesicles. rHMGB1 (25 ng/mL) was incubated with CpG-ODN-Cy5 (1018, 5 μg/mL) for 60 minutes and added to BMDMs treated with 20 ng/mL LMB for 45 minutes. ▴ indicate TLR9 extensively colocalized with CpG-DNA/HMGB1-containing vesicles. (E) Addition of rHMGB1 increases colocalization of HMGB1 with TLR9. Different amounts of rHMGB1 as indicated were incubated with wt and HMGB1-deficient macrophages (IFLMDs) for 10 minutes. Colocalization of TLR9 with HMGB1 was determined. (F) Translocation of HMGB1 into ERGIC-53–containing vesicles can be blocked by LMB and restored by exogenous rHMGB1. BMDMs were starved for 3 hours and then treated with LMB or left untreated for 60 minutes. Cells were incubated with CpG-ODN (10 μg/mL) or rHMBG1 (50 ng/mL) for 10 minutes in the presence or absence of LMB.