BMDMs contain vesicles rich in HMGB1 and TLR9, which participate in CpG-ODN recognition. (A) HMGB1 is associated with TLR9 prior to CpG-ODN treatment. Lysates from TLR9-deficient and wt splenocytes were used to identify the TLR9 band (left panel). WEHI-231 cells were treated with CpG-ODN (10 μg/mL), GpG-ODN (10 μg/mL), or peptidoglycan (PGN, 10 μg/mL) for the times indicated. HMGB1 was immunoprecipitated from cell lysates, and the presence of TLR9 or HMGB1 in the precipitated materials was detected by IB (left panel). Wt and TLR9-deficient splenocytes were treated with CpG-ODN (10 μg/mL) for the indicated time points or left untreated (top right panel). TLR9 was immunoprecipitated, and coprecipitation of HMGB1 with TLR9 was determined by IB. Whole-cell lysates from wt, HMGB1-deficient, and TLR9-deficient cells were probed by anti-HMGB1 and anti-TLR9 antibodies (bottom right panel). (B) Confocal microscopy of quiescent BMDMs reveals that HMGB1 and TLR9 colocalize in vesicular structures (indicated by triangles in z-stack images), which consistently appear close to the nucleus. Depth sections across 2 vesicles are shown in i-iv. Proteins were detected with anti-HMGB1/FITC and anti-TLR9/rhodamine. (C) BMDMs treated with CpG-ODN-Cy5 (1018, 5 μg/mL) as indicated were fixed, permeabilized, and stained with anti-TLR9/rhodamine and anti-HMGB1/FITC. △ indicate HMGB1/TLR9-containing vesicles; ▴ indicate CpG-DNA–containing vesicles. Solid arrows point to dispersed CpG-ODN-Cy5 staining in the tubular lysosomal compartment. (D) Quantification of the percentage of BMDMs containing at least one TLR9/HMGB1 vesicle. Cells were treated or not with CpG-ODN (1018, 5 μg/mL) in at least 3 independent experiments. Vesicles clearly visible within a single plane were counted.