FANCC suppresses TNF production and NF-κB activation induced by TLR8 ligands. Results in THP-1 cells are from 1 of 4 identical experiments. THP-1 Blue cells transduced with control (not targeted) shRNA or with shRNA targeting FANCC were incubated 24 hours with various concentrations of R-848. Expression of the NF-κB reporter was quantified colorimetrically (A), and TNF-α production was quantified by the use of ELISA (B). Dose-response curves shown are representative of 4 (A) and 2 (B) assays. SEAP and TNF-α responses of untransduced cells matched precisely those of cells transduced with the control shRNA (not shown). The specific lentiviral shRNA targeting FANCC (shFANCC) was selected based upon its capacity to induce MMC hypersensitivity and suppress monoubiqutinylation of FANCD2 in THP1 Blue cells (data are shown in supplemental Figure 5). (C) TNF-α release from primary murine splenic cd11b⁺ macrophages exposed for 24 hours to multiple doses of R848 showed a 4-fold increase in TNF-α released in cultures of Fancc^−/− macrophages. Macrophage-depleted mononuclear cells produced no detectable TNF-α either before or after exposure to R848. Error bars represent mean (± SD).