Apocynin

DCs (2·10⁶ cells/ml) were stimulated with UV-treated USA300 at MOI 10, unless otherwise stated, and incubated at 37°C in 5% CO₂ at indicated hours. DCs were treated with 100 µg/ml mannan from Saccharomyces cerevisiae (Sigma-Aldrich, St. Louis, MO, USA) 30 min prior to bacteria stimulation. Prior to bacterial stimulation BMDCs were pre-treated with 0.5 µg/ml cytochalasin D (CytD) (Sigma-Aldrich) for 1 h to inhibit bacterial uptake, with 300 µM apocynin (R&D systems, Minneapolis, MN, USA) for 30 min to inhibit assembly of the NADPH oxidase and ROS production and with 50 nM bafilomycin A1 (BafA) (Sigma-Aldrich) or 40 mM NH₄Cl (Sigma-Aldrich) for 1 h to inhibit phagosome acidification. MAPK pathways were inhibited in DCs by pre-treating for 1 h with 25 µM JNK inhibitor (Sigma-Aldrich, SP600125), 10 µM p38 inhibitor (Sigma-Aldrich, SB203580) or 10 µM MEK 1/2 inhibitor (Sigma-Aldrich, UO126). Factors present during inflammation such as GM-CSF (15 ng/ml) or IFN-γ (1 ng/ml, Thermo Fisher Scientific) was added to the medium of DCs just prior to bacterial stimulation.

Syk inhibitor (ER 27319 maleate), TLR2 inhibitor (human TLR2 mAb, clone 383936), isotype control for TLR2 mAb (mouse IgG2B, clone 20116), pan-caspase inhibitor (Z-VAD-FMK), caspase-8 inhibitor (Z-IETD-FMK), NADPH-oxidase inhibitor (apocynin) and phagocytosis inhibitor (cytochalasin D) were purchased from R&D Systems. TLR4 inhibitor (CLI-095) was from Invivogen, IRAK-1/4 inhibitor from EMD Millipore, glycine from Sigma-Aldrich. All inhibitors were used at concentrations indicated in the figure legends. All reagents were reconstituted in sterile dimethyl sulfoxide except for the antibodies that were dissolved in PBS and glycine that was dissolved in distilled water.