STING ligands activate the noncanonical NF-κB signaling pathway in a STING-dependent manner in MEFs. (A) STING^+/+ and STING^−/− MEFs were stimulated with 10 μg/ml of dsDNA90 or 10 μg/ml of poly(I·C) for the indicated times. The expression levels of STING, NF-κBp52, NF-κBp100 phosphorylated at Ser866/870 (p-NF-κBp100), and β-actin were determined by immunoblotting. (B) Immortalized MEFs derived from wild-type (WT) and IKKα-deficient (IKKα^−/−) mice (top panels) or wild-type (TBK1^+/+) and TBK1-deficient (TBK1^−/−) mice (bottom panels) were stimulated with 10 μg/ml of dsDNA90 for the indicated times. The expression levels of IKKα, TBK1, NF-κBp52, NF-κBp100 phosphorylated at Ser866/870 (p-NF-κBp100), and β-actin were determined by immunoblotting. The phosphorylation state of NF-κBp100 is indicated by an arrowhead. (C) Model of dsDNA-mediated canonical and noncanonical NF-κB activation triggered by STING. Upon dsDNA stimulation, STING is activated and traffics with TBK1 as a signaling complex from the ER to a perinuclear endosomal compartment to activate IRF3 (right arm) and NF-κB. STING is also activated by cGAMP produced by cGAS, which was recently identified as a candidate DNA sensor. TRAF6 may be recruited to the signaling complexes with STING and TBK1, which in turn activates the canonical NF-κBp65 signaling pathway through the IKKαβ activation loop (center arm). STING-mediated NF-κBp65 activation may also contribute to dsDNA-mediated IFN-β production. On the other hand, STING may also activate the noncanonical NF-κB signaling pathway through the TRAF3-IKKα axis, leading to modulation of the dsDNA-mediated canonical NF-κB signaling pathway (left arm). Thus, TRAF3 may be involved in the modulation of canonical and noncanonical dsDNA-mediated NF-κB activation triggered upon STING activation.