The HMGB1/TLR9-containing vesicles colocalize with calnexin, GM130, and ERGIC-53, but lack lysosomal markers. (A) TLR9-containing vesicles accumulated the acidophilic fluorophore LysoTracker. BMDMs were treated with LysoTracker for 30 minutes prior to stimulation with CpG-ODNs (5 μg/mL), then fixed, permeabilized, and stained with anti-TLR9/FITC. Open triangles indicate representative vesicles. (B) BMDMs were stained with anti-HMGB1/FITC and anti-LAMP-1/rhodamine following stimulation with CpG-ODNs (5 μg/mL). △ indicate HMGB1-containing vesicles; ▴, CpG-DNA-containing vesicles; and ↑, tubular lysosomal compartment. (C-D) BMDMs were stained with anti-GM130/Alexa488 (C) or anti-calnexin/Alexa488 (D) and anti-TLR9/Alexa568 or anti-HMGB1/Alexa568, prior to or following stimulation with CpG-ODN-Cy5 (5 μg/mL) for 10 minutes. Confocal images were acquired by indirect immunofluorescence. Solid arrows indicate colocalization between GM130/calnexin and TLR9 or GM130 and HMGB1. Solid triangles indicate vesicles containing CpG-ODN-Cy5 as well as GM130 and TLR9 or HMGB1. Open triangles indicate vesicles containing HMGB1/TLR9 and CpG-ODN but lack calnexin or GM130. (E) Control staining of BMDMs with antimouse-Alexa568/antirabbit-Alexa568 and antigoat-Alexa488. (F) Percentages of vesicles containing CpG-ODNs alone, CpG-ODN in the presence of TLR9/HMGB1, or CpG-ODN in the presence of TLR9/HMGB1 and GM130 or calnexin. Vesicles were analyzed from 2 independent experiments. (G,H) Quiescent (G) or treated (H) BMDMs were stained with anti-ERGIC-53/Alexa488 and either anti-TLR9/Alexa568 or anti-HMGB1/Alexa568. Rectangular regions showing representative colocalization between ERGIC-53/TLR9 and ERGIC/HMGB1 (solid arrows) are magnified. Colocalization of CpG-ODNs with TLR9/HMGB1 in the presence or absence of ERGIC-53 is shown with solid or open triangles, respectively.