Inhibition of the TLR8 pathway suppresses R848 activation of NF-κB in FANCC-deficient cells. THP-1 Blue cells expressing either control (nontargeted shRNA) or FANCC shRNA (target and shRNA sequences are shown in supplemental Table 4) were pretreated for 2 hours with 25μM IKK inhibitor (A) or 25μM IRAK-1/4 inhibitor (B) and then incubated for 24 hours with 30μM R-848. Both inhibitors reduced NF-κB activation in THP-1 Blue cells exposed to R-848. (C) Stable expression of TLR8 shRNA (target and shRNA sequences are shown in supplemental Table 4) in THP-1 Blue cells reduced TLR8 mRNA levels by 73%. Nontargeted shRNA had no effect on TNF-α mRNA. (D) Stable coexpression of TLR8 shRNA with either control (nontarget) or FANCC shRNAs lowered R-848–induced NF-κB activation. Specificity of TLR8 shRNA was confirmed by in experiments that showed that this shRNA did not suppress LPS-induced NF-κB activity (not shown). Error bars represent mean (± SD). (E) NF-κB activation (assessed by quantification of SEAP, y-axis) was quantified in the ground state and in cells treated with R848 in the presence and absence of SB203580, a p38 MAPK inhibitor. The inhibitor suppressed the R848 response in both control and shFANCC cells. (F) TNF-α production was likewise suppressed by SB203580 in THP1 Blue shFANCC cells treated with R848. Control cells not treated with R848 did not produce TNF-α. (G) Likewise, high-level endogenous TNF-α production in FANCC-deficient HSC536 lymphoblasts (control) was also suppressed by SB203580. HSC536/FANCC cells are isogenic FA-C lymphoblasts expressing wild-type FANCC cDNA.