Gene silencing on CD14+ monocytes was performed as previously published (112) with some modifications. Briefly, monocytes were resuspended at 1.5 × 106 cells/ml in complete medium and transfected with 200 mM small interfering RNA (siGENOME SMARTpool, Dharmacon) for TLR7, TLR8, ATF1, ATF2, FOSL1, or FOSL2 using HiPerFect (QIAGEN). 36 hours after transfection, some cells were lysed to confirm target gene silencing, and the rest of monocytes were harvested, plated at 5 × 104 cells per well in 96-well plates and stimulated with IMQ or ssRNA40 at 5 μg/ml for 16 hours. Supernatant was collected for ELISA determination of cytokines, and cells were lysed with RLT Plus buffer (QIAGEN) for gene expression analysis.

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