STING is an essential mediator of the Ku70-mediated production … · 293T cells (Fig. 3C). We next investigated whether the removal of STING from HEK 293 cellswould modifythe DNA-stimulated

SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

IMMUNOLOGY

1Laboratory of Human Retrovirology and Immunoinformatics, Applied and Develop-mental Research Directorate, Leidos Biomedical Research Inc., Frederick NationalLaboratory for Cancer Research, Frederick,MD21702, USA. 2Laboratory of Proteomicsand Analytical Technologies, Cancer Research Technology Program, Leidos BiomedicalResearch Inc., Frederick National Laboratory for Cancer Research, Frederick, MD21702, USA. 3Laboratory of Immunoregulation, National Institute of Allergy andInfectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.*Present address: Inova Schar Cancer Institute, Inova Health System, Annandale,VA 22003, USA.†Corresponding author. Email: [email protected]

Sui et al., Sci. Signal. 10, eaah5054 (2017) 18 July 2017

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STING is an essential mediator of the Ku70-mediatedproduction of IFN-l1 in response to exogenous DNAHongyan Sui,1 Ming Zhou,2* Hiromi Imamichi,3 Xiaoli Jiao,1 Brad T. Sherman,1

H. Clifford Lane,3 Tomozumi Imamichi1†

We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that inducesthe production of interferon-l1 (IFN-l1) by human primary cells and cell lines. IFN-l1 is a type III IFN and hassimilar antiviral activity to that of the type I IFNs (IFN-a and IFN-b). We observed that human embryonic kidney(HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did notproduce IFN-l1 in response to DNA unless they were reconstituted with STING. Conversely, parental HEK 293 cellsproduced IFN-l1 after they were exposed to exogenous DNA; however, when STING was knocked out in the HEK293 cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editingsystem, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was locatedin thenucleus and then translocated to the cytoplasmuponDNAexposure to forma complexwith STING.Additionally,the DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING inprimary human macrophages inhibited their ability to produce IFN-l1 in response to transfection with DNA or infec-tion with the DNA virus HSV-2 (herpes simplex virus–2). Together, these data suggest that STING mediates the Ku70-mediated IFN-l1 innate immune response to exogenous DNA or DNA virus infection.

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INTRODUCTIONImmune recognition of pathogens is mediated by germ line–encodedpattern recognition receptors (PRRs), which recognize conserved mi-crobial structures termed pathogen-associated molecular patterns(PAMPs) (1, 2). PRR families include the Toll-like receptors (TLRs),the retinoic acid–inducible gene I (RIG-I)–like receptors, and a diversefamily of cytosolic DNA sensors (3–7). Once engaged by PAMPs, PRRsstimulate intracellular signaling pathways and activate transcriptionfactors, such as nuclear factor kB and interferon (IFN) regulatory factor3 (IRF3), which lead to the increased production of proinflammatorycytokines, such as tumor necrosis factor–a and interleukin-1b (IL-1b),and of the antiviral type I IFNs, IFN-a or IFN-b (8, 9).

Considerable effort has beenmade to try to elucidate the initial type IIFN signaling events that enable cells to detect the presence of cytosolicDNA. This search has led to the identification ofmultiple DNA sensors.The IFN-inducible protein DAI (DNA-dependent activator of IRFs)was the first protein reported as a potential mediator of the IFN responseto cytosolicDNA(10). LRRFIP1 (leucine-rich repeat flightless-interactingprotein 1) and ABCF1 (ATP-binding cassette, subfamily F member 1)also bind to DNA directly and stimulate IFN responses (11, 12). TheAIM2 (absent in melanoma 2)–like proteins, including human IFI16(IFN-g–inducible protein 16) and its mouse ortholog protein p204,are also implicated in DNA-mediated IFN responses (13–15). More-over, some DExD/H-box helicases, including DDX41 (DEAD-box heli-case 41),DHX9 (DExH-boxhelicase 9), andDHX36 (DExH-boxhelicase36), are also postulated to act as sensors of cytosolic DNA (16–18). Last,some proteins with known functions in DNA damage responses are

mediators of the antiviral response triggered by cytosolic DNA. Theseinclude components of the DNA-PK (DNA-dependent protein kinase)complex, which is composed of Ku70, Ku80, and DNA-PKcs as well asMRE11 (meiotic recombination 11 homolog A) (19, 20).

Type III IFNs are lesswell-characterized IFNswith a similar antiviralactivity to that of the type I IFNs, and they include IFN-l1, IFN-l2, andIFN-l3 (also known as IL-29, IL-28A, and IL-28B, respectively) as wellas IFN-l4 (21–26). Expression of the genes that encode IFN-l proteinsis inducible by infection with many types of viruses (27–29). However,analysis of the murine genome showed that the gene encoding themouse ortholog of human IFN-l1 lacks exon 2 entirely and containsa stop codon within exon 1 even in wild mice; thus, the Ifnl1 gene doesnot encode a functional IFN-l1 protein (30). Therefore, any investiga-tion of the physiological relevance of IFN-l1 in a mouse model isrestricted. Compared to the signaling pathways associated with the pro-duction of type I IFNs, the signaling pathways underlying the produc-tion of type III IFNs are poorly understood.Wepreviously identified theDNA-PK component Ku70 as a DNA sensor that stimulates type IIIIFN production by human primary macrophages and human cell lines.Ku70, initially characterized as a DNA repair protein, specifically bindsto cytosolic DNA (such as after transfection) or viral DNA and thenactivates the IFN transcription factors IRF1 and IRF7, leading to robustproduction of IFN-l1 (31).Our previous study indicated that the induc-tion of type III IFN production is distinct from that of type I IFN pro-duction; however, the identity of the adaptor protein downstream ofKu70was unknown.We also reported that SV40T-antigen–transformedhuman embryonic kidney (HEK) 293 cells (herein referred to as 293Tcells) donot produce IFN-l1 in response toDNAstimulation.Themech-anism underlying this lack of response is unknown (32).

The adaptor protein STING (stimulator of IFN genes) was reportedto play a pivotal role in responding toDNAbymediatingTBK1 (TANKbinding kinase 1)–dependent activation of IRF3 in response to cytosolic,double-stranded DNA (33–35). A wealth of information on the role ofSTING inDNA sensing and on themechanisms whereby it contributesto signaling in the induction of type I IFN production has beendiscovered. In the signaling pathway to produce type I IFNs, STINGacts

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downstream of several cytosolic DNA sensors, including DAI, DDX41,cGAS [cyclic GMP (guanosine monophosphate)–AMP (adenosinemonophosphate) synthase], and IFI16 (10, 15, 17, 36). Other mediatorsinvolved in orchestration of the innate immune response includeb-catenin, which functions downstream of LRRFIP1 to stimulateIFN production, and the adaptor protein MyD88 (myeloid differenti-ation primary response 88), which is involved in responding to cyto-solic DNA through DHX9- or DHX36-mediated innate immuneresponses (11, 16, 18). Here, we have looked for host factor(s) involvedin the Ku70-mediated induction of type III IFN production in re-sponse to exogenous DNA. Our findings offer a new perspective onDNA sensing and have implications for host defense, vaccine devel-opment, and autoimmunity.

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RESULTSAs compared to HEK 293 cells, 293T cells do not produceIFN-l1 in response to DNAWe previously reported that Ku70 is a cytosolic DNA sensor that in-duces IFN-l1 production by primary human macrophages and somenonimmune cells, such as HEK 293 cells and human rhabdo-myosarcoma cells (31), but not by 293T cells (32). Here, we transfected293T cells and HEK 293 cells with plasmid encoding green fluorescentprotein (GFP). One day later, the extent of transfection was monitoredby fluorescence microscopy analysis. Similar amounts of GFP were de-tected in both cell lines (Fig. 1A). To determine the extent of expressionof the genes encoding IFNA, IFNB, IFNL1, and IFNL2/3 in response tothe transfection, we extracted cellular RNA from the transfected cellsand measured the abundances of the appropriate mRNAs with areal-time reverse transcription polymerase chain reaction (RT-PCR) as-say. Consistent with our previous report (32), HEK 293 cells showed amore than 200-fold increase in the abundance of IFNL1 mRNA aftertransfection with DNA. However, the abundances of IFNA, IFNB,and IFNL2/3mRNAs were only 1.3-, 3.6-, and 61.3-fold greater, respec-tively, compared to those in untreated cells (Fig. 1B). In contrast, none ofthese mRNAs were increased in abundance in 293T cells after transfec-tion with DNA (Fig. 1B). To further confirm induction of the IFN re-sponse, we used enzyme-linked immunosorbent assays (ELISAs) tomeasure the amounts of IFN-l1, IFN-a, IFN-b, and IFN-l2/3 proteinsproduced by the cells. IFN-l1 was the major IFN produced by the HEK293 cells, which had concentrations of 1393 ± 43.1 pg/ml in the culturemedium, whereas the concentrations of IFN-l2/3, IFN-a, and IFN-bwere reduced at 371.0 ± 0.9, 61.3 ± 0.6, and 157.3 ± 1.9 pg/ml, respectively(Fig. 1C).Consistentwith the gene expressiondata,we failed to detect anyIFN-a, IFN-b, IFN-l1, or IFN-l2/3 production by the transfected 293Tcells (Fig. 1C). Additionally, a time course of the production of IFN-a,IFN-b, IFN-l1, and IFN-l2/3 wasmeasured in the transfectedHEK cells(fig. S1). On the basis of these data, we hypothesized that one or moreimportant signaling factors were missing in the 293T cells.

293T cells lack endogenous STINGSTING is an importantmediator in severalDNA-sensing pathways thatare associatedwith the induction of type I IFN responses (10, 15, 17, 36).To determine whether differences in the amounts of STING or Ku70might be associated with the differential ability of 293T and HEK 293cells to produce IFN-l1, we compared the amounts of STINGandKu70mRNAandprotein between the two cell lines. The abundance of STINGmRNAwas less in 293T cells than inHEK293 cells (Fig. 2A). Consistentwith this result, the abundance of STINGprotein, asmeasured byWest-


ern blotting analysis, was alsomarkedly reduced in 293T cells comparedto that in HEK 293 cells. There was no substantial difference in theamount of Ku70 protein between 293T and HEK 293 cells (Fig. 2B).As additional controls, we also assessed the amounts of b-catenin andMyD88 proteins in 293T andHEK 293 cells; however, they were similarin both cases (fig. S2).

STING is an essential mediator in Ku70-mediated inductionof IFN-l1 production in response to DNATo test the hypothesis that the absence of STING in 293T cells wasresponsible for their inability to produce IFN-l1 in response to DNA

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Fig. 1. Transfection of 293T cells with GFP-encoding plasmid DNA does not in-duce the production of IFN-a, IFN-b, IFN-l1, or IFN-l2/3mRNA and protein. (A toC) HEK 293 and 293T cells were transfected with plasmid pCMV-GFP. (A) Twenty-fourhours later, the cells were observed under a fluorescence microscope. Green fluores-cence was shown in the context of total cells. Images are representative of threeindependent experiments. (B) Total RNA was extracted and the relative amounts ofIFNLA, IFNB, IFNL1, and IFNL2/3mRNAsweremeasuredby real-timeRT-PCR. The relativeexpression of the indicated genes was compared to that in untreated cells. Data aremeans ± SDof three independent experiments. (C) Forty-eight hours after transfection,the cell culturemediumwas collected, and the concentrations of the indicatedproteinswere measured by ELISA. Data are means ± SD of three independent experiments.

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exposure, we established an IFN-l1 reporter assay system. In this sys-tem, 293T cells were cotransfected with pGL4–IFN-l1, a reporter plas-mid containing the IFNL1 promoter region, and different amounts ofplasmid encoding Ku70 with or without 100 ng of plasmid encodingSTING. To fully induce Ku70 and STING, the transfected cells wereincubated for 48 hours andwere then further stimulated by being trans-fected with noncoding plasmidDNA, and IFNL1 promoter activity wasquantitated by relative luciferase activity (Fig. 3A).We found that Ku70induced IFNL1 promoter activity only in the presence of STING, and itdid so in a concentration-dependentmanner. Note that Ku70 alonewasnot able to induce IFNL1 promoter activity. We detected substantialamounts of STING in those 293T cells that were transfected with theSTING-encoding plasmid (Fig. 3B); however, STING was undetectablein 293T cells that were transfected with an empty vector. To furtherconfirm the effect of exogenous STING in 293T cells on IFNL1 expres-sion, we performed real-timeRT-PCR assay using cellular RNA isolatedfrom DNA-stimulated, STING-overexpressing 293T cells. The abun-dance of IFNL1 mRNA in the STING-expressing 293T cells wasincreased in response to DNA stimulation (Fig. 3C), whereas 293T cellsfailed to induce IFNL1 expression if theywere transfectedwith an emptyplasmid,whichwas similar to the IFNL1 response observed in untreated293T cells (Fig. 3C). We next investigated whether the removal ofSTING from HEK 293 cells would modify the DNA-stimulated induc-tion of IFNL1 expression. For these experiments, STING knockout(STING KO) HEK 293 cells were generated through the clustered reg-


ularly interspaced short palindromic repeats (CRISPR)/Cas9 genomeediting system. STING was undetectable in the STING KO cells; how-ever, it was detectable after the cells were transfected with a STING ex-pression plasmid (Fig. 3D). The abundance of Ku70was similar in all ofthe cell lines examined, suggesting that the loss of STING had no effecton Ku70 abundance. STINGKO cells did not exhibit IFNL1 expressionafter transfection with DNA. However, the DNA-stimulated inductionof IFNL1 expression was restored in cells transfected with the STING-encoding plasmid (Fig. 3E). Together, the results suggest that STING isan essential mediator of the DNA-stimulated induction of IFNL1.

To exclude the possibility that the role of STING in facilitatingIFN-l1production in response toDNAwas cell type–dependent,weper-formed similar experiments with the THP-1 cell line. THP-1 cells arehuman leukemia monocytic cells, which have been extensively used tostudymonocyte andmacrophage functions, mechanisms, and signalingpathways as well as nutrient and drug transport (37). Western blottinganalysis confirmed that THP-1 cells have endogenous Ku70 andSTING. Transfection of the cells with small interfering RNAs (siRNAs)specific for Ku70 and STING reduced the abundances of the target pro-teins by 51 and 66%, respectively, compared to those in cells transfectedwith control siRNA (Fig. 3F). Using the cells in which Ku70 or STINGwere knocked down, we further performed transfections with plasmidDNA and assessed the expression of IFN-l1. Similar to HEK 293 cells,THP-1 cells exhibited a substantial (12,131-fold) increase in IFNL1mRNA in response to transfection with DNA; however, this inductionwas inhibited in cells in which the abundances of either Ku70 or STINGwere knocked downby 59 and 78%, respectively (Fig. 3G). In cells trans-fected with both siRNA-Ku70 and siRNA-STING, the induction ofIFNL1 expression was inhibited by 88% compared with that in the cellsthat were not exposed to siRNA (Fig. 3G). Consistent with these data,the abundance of IFN-l1 protein in the THP-1 cells in which the abun-dance of Ku70 and STINGwas reduced by 96% compared to that in thecells that were not exposed to siRNA (Fig. 3H), suggesting that theKu70-STING pathway plays a role in the DNA-stimulated inductionof IFN-l1 production in THP-1 cells (Fig. 3, G andH). The knockdownof Ku70 and STING in the THP-1 cells also suppressed the productionof IFN-a, IFN-b, and IFN-l2/3 (fig. S3).

We previously reported that cross-talk between signaling by theDNAsensor IFI16 and the RNA sensor RIG-I mediates the type III IFN re-sponse. Here, we investigated whether IFI16 was also involved in theKu70-mediated production of IFN-l1 in response to exogenous DNA.We knocked down IFI16 by transfecting the cells with IFI16-specificsiRNA and confirmed the efficiency of knockdown byWestern blottinganalysis (fig. S4A). However, the extent of IFNL1 induction was notchanged in the IFI16-knockdown cells compared with that in the cellsthat were not treated with siRNA (fig. S4B).

Ku70 translocates from the nucleus to the cytoplasm andforms a complex with STING in response to DNAOur findings led us to investigate the physical relationship betweenSTING and Ku70. The locations of endogenous Ku70 and STINGwereanalyzed by immunofluorescence staining and confocal microscopyanalysis of unstimulated and DNA-stimulated cells (Fig. 4A). Thecellular localization was verified by counterstaining nuclei with 4′,6-diamidino-2-phenylindole (DAPI). In unstimulated cells, Ku70 wasdetected in the nucleus, whereas STING was found in the cytoplasm(Fig. 4A, top). After the cells were stimulatedwithDNA,we found thatKu70 translocated from the nucleus to the cytoplasm, whereas STINGremained in the cytoplasm (Fig. 4A, bottom). This translocation of

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Fig. 2. Comparison of the amounts of endogenous STING and Ku70 betweenHEK 293 cells and 293T cells. (A) Total RNA was extracted from HEK 293 and 293Tcells, and the relative amounts of STING and KU70mRNAs weremeasured by real-timeRT-PCR. The amounts of the indicated mRNAs were normalized to that of GAPDHmRNA. Data are means ± SD of three independent experiments. (B) Whole-cell lysatesof HEK 293 and 293T cells were analyzed by Western blotting with antibodies againstSTING and Ku70. b-Actin was used as a loading control. Western blots are representa-tive of three independent experiments.

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Ku70 from the nucleus to the cytoplasm provided an opportunity forSTING and Ku70 to interact with each other.

We further designed a coimmunoprecipitation assay to confirmwhether STING interacted with Ku70 in the context of stimulation withDNA. The results demonstrated that in the absence of exogenousDNA,FLAG-tagged Ku70 did not interact with exogenous STING. However,Flag-tagged Ku70 coimmunoprecipitated with STING when the cellswere treated with DNA (Fig. 4B). We also performed the coimmuno-precipitation assay with an anti-Myc antibody to pull down STING(Fig. 4C). The results of these experiments consistently supported thehypothesis that STING and Ku70 are found in the same complex onlyafter the cells are transfected with DNA (Fig. 4, B and C).


The DNA binding domain of Ku70 is required for Ku70 toform a complex containing STINGKu70 is composed of an a/b domain (I), a b-barrel DNA binding do-main (II), a linker (III), and a C-terminal domain (IV) (Fig. 5A). Toanalyze which domain is involved in the binding of Ku70 to STING,we constructed several truncated, Flag-tagged Ku70 mutants, contain-ing the I, II, and III domains (I.II.III), the I and II domains (I.II), orthe I domain alone (I). We then performed coimmunoprecipitation as-says with 293T cells transfected with plasmids encoding the Flag-taggedKu70mutants and detectedwhich of these bound to STING in responseto DNA.We found that Ku70 mutants with I.II.III.IV, I.II.III, I.II, or II.III.IV coimmunoprecipitated with STING; however, those Ku70 mu-tants with I.III.IV, I.III, I, or III.IV did not (Fig. 5B). Thus, it appearedthat theDNAbinding domain (II) was required for the binding of Ku70to STING. To confirm this finding, we constructed a Ku70mutant con-sisting of only the DNA binding domain (Ku70 mutant II). We foundthat this protein domain coimmunoprecipitated with STING (Fig. 5B),confirming the importance of this region of Ku70 in binding to STING.

A heterodimer of Ku proteins (Ku70 or Ku80) and the catalytic sub-unit DNA-PKcs are the components of the DNA-PK complex. We ad-ditionally confirmed whether Ku80 andDNA-PKcs were present in the

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Fig. 3. STING is critical for the DNA-stimulated, Ku70-mediated induction ofIFN-l1 production. (A) 293T cells were cotransfected with an IFNL1 reporter plasmid(pGL4–IFN-l1), a Renilla luciferase plasmid (pRL-TK), and different amounts of theKu70 expression plasmidwith or without transfectionwith 100 ng of the STINGexpres-sion plasmid, as indicated. Forty-eight hours after transfection, the cellswere then stim-ulated with 1 mg of noncoding plasmid DNA. Twenty-four hours after stimulation, thecells were collected for luciferase assays. Data are means ± SD of three independentexperiments. (B) Western blotting analysis was performed to confirm the presence ofSTING in the transfected 293T cells. Untreated 293T cells and 293T cells transfectedwith empty plasmid were included as controls. Whole-cell lysates were analyzed withanti-STING and anti-Ku70 antibodies. b-Actin was detected as a loading control. West-ern blots are representative of three independent experiments. (C) 293T cells were leftuntreated or were transfected with either an empty plasmid or a STING expressionplasmid and then were stimulated with 1 mg of plasmid DNA. One day later, IFNL1mRNA abundances were measured by real-time RT-PCR. Relative values weredetermined by comparison with untreated 293T cells. Data are means ± SD of threeindependent experiments. **P < 0.001. (D) Western blotting analysis was performed toconfirm the knockout of STING in the STING KO cells. Whole-cell lysates were analyzedwith anti-STING and anti-Ku70 antibodies. b-Actin was measured as a loading control.Western blots are representative of three independent experiments. (E) STING KO cellswere transfectedwith an empty vector or a STINGexpression vector, and the cells werethen stimulated by transfection with 1 mg of noncoding plasmid DNA. One day later,total cellular RNA was extracted and the relative abundance of IFNL1 mRNA wasmeasured. The values were compared to the amount of IFNL1mRNA in HEK cells thatwere transfected with empty vector without additional DNA stimulation. Data aremeans ± SD of three independent experiments. **P < 0.001. (F) Western blottinganalysis was performed to confirm the knockdown of Ku70 or STING in THP-1 cells.THP-1 cells were transfected with or without the indicated siRNAs and then weretransfected with DNA 1 day later. Whole-cell lysates were collected and incubatedwith anti-STING and anti-Ku70 antibodies. b-Actin was used as a loading control.Western blots are representative of three independent experiments. (G) Twenty-fourhours after the indicated THP-1 cells were stimulated by transfection with DNA, theabundance of IFNL1mRNA was measured by real-time RT-PCR. Relative values weredetermined by comparison with untreated THP-1 cells. Data are means ± SD of threeindependent experiments. (H) Forty-eight hours after the indicated THP-1 cells werestimulated by transfection with DNA, the cell culture medium was collected and theamounts of IFN-l1 were determined by ELISA. Data are means ± SD of threeindependent experiments.

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Ku70-STING complex. The proteins that coimmunoprecipitated withthe DNA binding domain of Ku70 were analyzed by Western blottingwith anti–DNA-PKcs, anti-STING, and anti-Ku80 antibodies, and theresult showed that Ku80was present in the complex but thatDNA-PKcswasnotnecessarily required for the interactionof theDNAbindingdomainof Ku70 with STING (fig. S5A). An IFNL1 promoter reporter assayfurther suggested that the DNA binding domain alone, like full-lengthKu70, dose-dependently activated IFNL1 promoter activity (fig. S5B).Ku80, even if present in the Ku70-STING complex, was not functionalin the DNA-mediated induction of IFN-l1 production (fig. S5B).

The activation of the transcription factors IRF3, IRF1, andIRF7 is required for the Ku70- and STING-mediatedproduction of IFN-l1 in response to exogenous DNAHaving identified STING as a critical protein in the Ku70-mediatedproduction of IFN-l1, we then determined which transcription factor(s)was involved. First, we tested the effect of knocking down IRF1, IRF3,or IRF7 on the induction of IFN-encoding genes.We transfectedHEK293 cells with siRNAs targeting IRF1, IRF3, or IRF7. On the following


day, the cells were then transfected withDNA. Whole-cell lysates were then col-lected and analyzed by Western blottingto determine IRF1, IRF3, and IRF7 abun-dance, and total RNA was extracted tomeasure the relative amounts of IFNmRNAs by real-time RT-PCR analysis.In the absence of exogenous DNA, theabundance of IRF3 in HEK 293 cellswas greater than that of either IRF1 orIRF7 (Fig. 6A). In response to transfectionwith DNA, the amounts of IRF1 andIRF7, but not that of IRF3, were increased(Fig. 6A). Transfection of the cells withIRF1-specific siRNA not only efficientlyreduced the abundance of IRF1 but alsodecreased that of IRF7 (Fig. 6A). In addi-tion, the abundances of both IRF7 andIRF1 were reduced in cells transfectedwith IRF7-specific siRNA. Furthermore,transfection of the cells with IRF3-specificsiRNAnot only reduced the abundance ofIRF3 but also reduced the amounts ofIRF1 and IRF7 (Fig. 6A). Our real-timeRT-PCR analysis showed that the induc-tion of IFNL1 expression and IFN-l1pro-tein production were inhibited in cells inwhich IRF1, IRF3, or IRF7 was knockeddown (Fig. 6, B andC). Similar resultswereshown for IFN-a, IFN-b, and IFN-l2/3(fig. S6, A and B).

Given that IRF3 and IRF7 reside in thecytosol and undergo serine phosphoryl-ation of their C-terminal regions in re-sponse to viral infection, which enablesthem to dimerize and translocate to thenucleus (38), we next evaluated the nucle-ar accumulation of IRF1, IRF3, and IRF7in HEK 293 cells and STING KO cells inresponse to stimulation DNA. We trans-

fected the cells withDNAand thenprepared nuclear fractions at varioustimes (Fig. 6D). Western blotting analysis showed that the abundancesof IRF1 and IRF7, but not that of IRF3, were increased in the nucleusof HEK 293 cells 48 hours after transfection. In contrast, no increase inthe nuclear accumulation of these transcription factors was observed inthe STINGKO cells (Fig. 6D). The results demonstrated that the DNA-dependent activation of IRF1 and IRF7 occurred only in the presence ofSTING.

To further elucidate the role of STING in the activation of IRF1 andIRF7 after transfection of cells with DNA, we used confocal microscopyto monitor the nuclear translocation of endogenous IRF1 and IRF7 inHEK 293 and STING KO cells (Fig. 6, E to H). Forty-eight hours aftertransfection of the cells with DNA, we observed substantial accumula-tion of both IRF1 and IRF7 in the nuclei of HEK 293 cells (Fig. 6, E andG). Similar changes were not observed in the STING KO cells (Fig. 6, Fand H). These results are consistent with STING playing a key role inactivating these transcription factors upstream of IFN gene induction.In summary, the absence of STING dampened the activation of IRF1and IRF7 and consequently resulted in reduced IFN-l1 production.

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Fig. 4. STING and Ku70 colocalize in the cytoplasm and form a protein-protein complex after cells are transfectedwith DNA. (A) Confocal microscopy analysis of the localization of Ku70 and STING in HEK 293 cells. HEK 293 cells weregrown on glass-coated, 35-mm culture dishes and mock-transfected or transfected with 1 mg of plasmid DNA. Twenty-four hours later, the cells were fixed and stained with anti-Ku70 (green) and anti-STING (red) antibodies. Nuclei werevisualized by DAPI (blue). Original magnification was ×60. Scale bars, 5 mm. Images are representative of threeindependent experiments. (B and C) 293T cells were transfected with the indicated combinations of plasmids encodingFlag-tagged Ku70 and Myc-tagged STING and were stimulated by transfection with DNA on day 2. On day 3, cytosoliclysates were subjected to immunoprecipitation (IP) with agarose conjugated with anti-Flag antibody (B) or anti-Mycantibody (C). Precipitatedproteinswere then analyzed byWesternblottingwith antibodies against the indicated targets.Input controls were also included. Western blots are representative of three independent experiments.

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Loss of STING inhibits IFN-l1 production by human primarymacrophages in response to transfection with DNA orinfection with herpes simplex virus–2To further demonstrate the physiologic relevance of STING in theKu70-mediated production of IFN-l1 in response to DNA or DNAvirus, we transduced human monocyte-derived macrophages (MDMs)with lentiviruses encoding short hairpin RNA (shRNA) targeting theSTING expression cassette. The transduced cells were then transfected


with DNA plasmids or infected with her-pes simplex virus–2 (HSV-2). Twenty-four or 48 hours later, the cells werecollected to analyze gene expression, andthe culture medium was harvested tomeasure the abundances of IFN proteins.We first showed that transduction withthe lentivirus led to a substantial reduc-tion in the abundance of STING protein;however, no changes in STING abun-dance were observed in untransducedcells or in cells transduced with lentivirusencoding control shRNA (Fig. 7A). Wefurther found that theDNA-increased ex-pression of IFNL1 was inhibited by up to80% when STING was knocked down byshRNA (Fig. 7B). Analysis of IFN-l1 pro-tein abundance in the cell culturemediumgave consistent results (Fig. 7C).

In addition, we also evaluated the effectof knocking down STING on IFN-l1 pro-duction in cells infected with HSV-2. Theresults of this experiment showed thatknocking down STING resulted in a re-duction in the amounts of IFNL1 mRNAandprotein (Fig. 7,DandE). Furthermore,knocking down STING also suppressedthe induction of IFNA, IFNB , andIFNL2/3 (fig. S7). Together, these data sug-gest that STING mediates the Ku70-dependent production of IFN in responseto exogenous DNA or infection with aDNA virus.

DISCUSSIONThe innate immune system is the firstline of defense against invading patho-gens. It is well known that microbial nu-cleic acids stimulate the production oftype I IFNs, such as IFN-a and IFN-b,as a key host defense strategy to limitthe replication of invading microorga-nisms (39). Many of these pathways andtheir capacity to induce type I IFN pro-duction have been extensively studied(40). Accumulated evidence suggests thatnon-self nucleic acids can also stimulatea type III IFN response (21, 23, 31, 32);however, the molecular mechanismsinvolved are poorly understood.

The DNA repair protein Ku70 also acts as a DNA sensor to inducethe production of IFN-l1, as opposed to IFN-a or IFN-b, in primarycells or cell lines, such as HEK 293 cells, but not 293T cells (31, 32).However, the downstream mediator of Ku70 was unclear. Here, bycomparing the properties of HEK 293 and 293T cells, we identifiedSTING, located downstream of Ku70, as an essential mediator of theproduction of IFN-l1 in response to exogenous DNA. The inabilityof 293T cells to produce IFN-l1 after transfection with DNA was

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Fig. 5. The DNA binding domain of Ku70 is essential for its ability to form a complex with STING. (A) Schematicrepresentation of the domain organization of Ku70. The molecular mass of each domain of Ku70 is labeled. WT, wild type.(B) 293T cells were transfected with plasmids encoding the indicated Flag-tagged truncated Ku70 constructs and Myc-tagged STING andwere then stimulated by transfectionwith DNA on day 2. One day later, cytosolic lysates were subjectedto immunoprecipitation with anti-Flag antibody–conjugated agarose. Precipitated proteins were analyzed by Westernblotting with anti-Flag and anti-STING antibodies. Input controls were also included. Western blots are representative ofthree independent experiments.

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Fig. 6. The activation of IRF1, IRF3, and IRF7involves the Ku70-mediated induction ofIFN-l1 production in response to exogenousDNA. (A) HEK 293 cells were left untreated orwere transfected with the indicated siRNAs.The cells were then transfected with DNA1 day later. Twenty-four hours after transfectionwith DNA, whole-cell lysates were analyzed byWestern blotting with antibodies against the in-dicated proteins. Untreated cells were includedas a control, and b-actin was used as a loadingcontrol for each condition.Western blots are rep-resentative of three independent experiments.(B) Total RNA was extracted 24 hours after DNAtransfection, and the relative abundance ofIFNL1 mRNA was measured by real-time RT-PCR. The relative values were determined bycomparison with untreated cells. Data aremeans ± SD of three independent experiments.(C) Forty-eight hours after DNA transfection, thecell culture medium was collected and theamounts of IFN-l1 protein were detected byELISA. Data are means ± SD of three inde-pendent experiments. (D) HEK 293 and STINGKO cells were stimulated by transfection with1 mg of plasmid DNA. Nuclear fractions of thecells were isolated at the indicated times aftertransfection and were analyzed by Westernblotting with antibodies against the indicatedproteins. NUP98 was measured as a loadingcontrol. Western blots are representative ofthree independent experiments. (E to H) HEK293 and STING KO cells were grown on glass-coated, 35-mm culture dishes. The cells weretransfected with plasmid DNA. Forty-eighthours later, the cells were fixed and stainedwithantibodies against IRF1 (green) (E and F) or IRF7(red) (G and H) and visualized by confocal mi-croscopy. Nuclei were visualized with DAPI(blue). Scale bars, 5 mm. Original magnificationwas ×60. Images are representative of threeindependent experiments.

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reversed by expression of the adaptor protein STING. In contrast, lossof STING inhibited the DNA-induced production of IFN-l1 by HEK293 cells. Additionally, we further clarified that this finding was notrestricted to HEK 293 cells by showing that the Ku70-STING pathwaywas required to mediate DNA-induced IFN-l1 production by THP-1cells and primary human macrophages.

Many studies about innate immune responses are performed withmice deficient in the protein of interest. However, in the case of IFN-l1,the mouse Ifnl1 gene lacks exon 2 and contains a stop codon withinexon 1 even in wild mice, which results in the failure to encode a func-tional IFN-l1 protein (30). Therefore, to demonstrate the physiologicalrelevance of STING in the Ku70-mediated production of IFN-l1 in re-sponse toDNA, we used a lentiviral approach to knock down STING inhuman primary macrophages. We found that the loss of STINGmark-edly inhibited the generation of IFNL1mRNA and protein in responseto transfection with DNA or infection with a DNA virus.


Confocal microscopy analysis showed that Ku70 was found in thenuclei of unstimulated cells. We found that Ku70 underwent a trans-location from the nucleus to the cytoplasm when the cells were trans-fected with a DNA plasmid. In contrast, STING was located in thecytoplasm in both unstimulated and stimulated cells. These results sug-gested that the translocation of Ku70 prompted an interaction betweenKu70 and STING. Further work is necessary to uncover the mecha-nisms about the translocation of Ku70. In addition, coimmunoprecipi-tation experiments demonstrated that Ku70 formed a complex withSTING in response to transfection with DNA. Ku70 is a componentof the heterotrimeric protein complex DNA-PK, which also containsKu80 and the catalytic subunit DNA-PKcs. DNA-PK acts as a PRR,binding to cytoplasmic DNA and stimulating the expression of genesencoding type I IFNs (19). This study also showed that Ku70 andSTING forma complex fromwhichKu70 dissociates 3 hours after stim-ulationwithDNA. Ferguson et al. (19) stated that the dissociation ofKu70and STING activated downstream signaling and induction of the ex-pression of genes encoding type I IFNs. Our data are complementaryto these findings in demonstrating that prolonged association of STINGandKu70may lead to the production of type III IFN as a late event afterexposure toDNA.Here, we focused on the late stage of the IFN immuneresponse.This time frame is consistentwith the timecourseof IFN-l1pro-duction in response to stimulation with DNA (fig. S1), which is alsoconsistent with our previous study (31). The result from ELISA demon-strated that there was a marked increase in the amount of IFN-l1 pro-tein secreted into the cell culture medium 24 to 48 hours aftertransfection. Furthermore, these experiments showed that detectableamounts of IFN-a, IFN-b, and IFN-l2/3 were found in the cell culturemedium but at a much lower abundance than that of IFN-l1 (fig. S1).Thus, the Ku70-mediated production of IFN-l1 in response to expo-sure to DNA or infection with a DNA virus is a dominant responsein HEK 293 cells, THP-1 cells, and primary human macrophages.

Ku70 is composed of fourmajor domains: thea/b domain, theDNAbinding domain (b-barrel), the linker domain, and the C-terminal do-main (41, 42). Here, we identified that the DNA binding domain wasessential for the binding of Ku70 to STING. Ku70 and Ku80 form het-erodimers, and the absence of one subunit destabilizes the other (43, 44).Both the Ku heterodimer (42) and DNA-PKcs (45) can bind directly toDNA; however, in the absence of either Ku70 or Ku80, the affinity ofDNA-PKcs for DNA is substantially reduced (46). These findings sug-gest that each component in the DNA-PK complex has an importantrole to play.However, we further found thatKu80, evenwhenpresent inthe Ku70-STING complex, was not functional in the DNA-mediatedinduction of IFN-l1 production (fig. S5). DNA-PKcs was not necessar-ily required for the interaction of the DNA binding domain of Ku70with STING (fig. S5A), whereas this DNA binding domain itself wasfunctional in the DNA-mediated induction of IFN-l1 production(fig. S5B). In summary, although Ku80 or DNA-PKcs was present inthe complex containing Ku70 and STING, they were not functionalin the induction of IFN-l1 production in response to DNA.

Previous studies identified the STING-TBK1-IRF3 pathway as play-ing an important role in the induction of type I IFN production in re-sponse to different forms of nucleic acids (47, 48). Here, we providedfurther evidence to show that the transcription factors IRF1, IRF7,and IRF3 were all involved in the DNA-dependent induction of IFN-l1 production. A major distinction between IRF3 on the one hand andIRF1 and IRF7 on the other is that IRF3 is constitutively found in mostcell types, whereas IRF1 and IRF7 are found in cells only after the ex-posure of cells to type I IFNs (49). Our data confirmed that production

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Fig. 7. Knocking down STING inhibits the production of IFN-l1by humanMDMstransfected with DNA or infected with HSV-2. (A to E) Human macrophages wereleft untreated, were mock-transduced, or were transduced with lentivirus expressingcontrol shRNA (Lenti-shCtrl) or shRNA targeting STING (Lenti-shSTING). (A) Seventy-twohours after transduction, the indicated cells were analyzed by Western blotting withantibody against STINGprotein.Western blots are representative of three independentexperiments. (B and C) The indicated transduced macrophages were transfected withlinearizedDNA. Total cellular RNAwas then analyzed by real-time RT-PCR to determinethe relative abundance of IFNL1mRNA (B), whereas cell culture medium was analyzedby ELISA to determine the amounts of IFN-l1 protein (C). Data aremeans ± SD of threeindependent experiments. (D and E) The indicated transduced macrophages wereinfected with HSV-2 at a multiplicity of infection (MOI) of 0.15. Total cellular RNA wasthen analyzedby real-timeRT-PCR todetermine the relative abundance of IFNL1mRNA(D), whereas cell culture medium was analyzed by ELISA to determine the amounts ofIFN-l1 protein (E). Data are means ± SD of three independent experiments.

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of the IRF1 and IRF7 proteins was induced in DNA-transfected cellsand was inhibited by knockdown of IRF3, indicating that IRF1 andIRF7 production required IRF3. Our data further showed that there wasa certain amount of IRF3 that accumulated in the nucleus, but the in-tensity of the signal did not show differences between DNA-stimulatedand unstimulated HEK 293 cells or STING KO cells. This finding sug-gests that IRF3 might be activated at an early stage but that IRF3 is notresponsible for the late-stage induction of IFN-l1 production. Thegenes encoding IRF1 and IRF7 were induced through the activationof IRF3 at an early stage and then contributed to the abundant induc-tion of IFN-l1 production at a later stage. This observation led us todivide the model of Ku70-mediated IFN-l1 production into two dis-tinct steps. First, the immediate or early response involved a very low(or undetectable) amount of IFN produced through the activation ofIRF3. Second, a low level of synthesis of IFNs from the first step resultsin positive autocrine feedback by the production of IRF1 and IRF7,which provides efficient amplification of IFN-l1 production at a laterstage (49). This two-stage model is consistent with the time course ofDNA-stimulated, Ku70-mediated IFNproduction (fig. S1). In cells trans-fectedwithDNA,wewere unable to detect the expression of IFNA, IFNB,or IFNL2/3 at earlier times (3 and 6 hours), and we detected IFN-l1 atlowabundance.Robust amounts of IFN-l1were produced at a later stage,that is, at 24 to 48 hours after the cells were stimulated with DNA. Thisresult further suggested that the production of IFN-a, IFN-b, and IFN-l2/3 was simultaneously induced together with that of IFN-l1. The sameinduction kinetics implied that the induction of production of these fourIFNs shared a similar signaling transduction pathway in response to exog-enous DNA, in which Ku70 and STING played an important role in thedominant induction of IFN-l1 production with the simultaneous induc-tion of IFN-a, IFN-b, and IFN-l2/3 production to a much lesser extent.

In addition, this Ku70-STING-IRF3-, IRF1-, and IRF7-mediatedpathway to induce IFN-l1 production differs from the IFI16-STING-IRF3–mediated IFN-l1 pathway, which we have previously reported(32), because IFI16-induced IFNL1 signaling peaked at 6 hours afterstimulationwithDNA stimulation, and IRF1 and IRF7 are not involved(32). Note that the exogenous DNA that we used for the induction ofIFN-l1 in the Ku70 pathway is distinct from theDNA that was used forthe stimulation of the IFI16 pathway. In our current study of the Ku70pathway in the induction of IFN-l1 production, linearized DNA andHSV-2 were used, whereas in the study of the IFI16-mediated pathwayfor the induction of IFN-l1 production, circular DNA andHSV-1 viruswere used (15, 32). The knockdown of IFI16 has no effect on the DNA-induced induction of IFN-l1, indicating that IFI16 is not involved in theKu70-mediated IFN-l1 induction pathway (fig. S4). Therefore, it is stillan interesting research area to discover the mechanism of distinct path-ogen recognition patterns in host cells in response to very similar for-eign invasion. Together, our results suggest that STING is an essentialmediator downstream of Ku70 for the DNA-induced production ofIFN-l1. This finding advances our understanding of the regulationof the innate immune response and expands the current repertoire ofDNA-sensing mechanisms. These findings may be of value in further-ing our understanding of the function of the immune system in healthand disease.

MATERIALS AND METHODSCells, antibodies, and virusesHEK 293 cells, 293T cells, and THP-1 cells were obtained from theAmerican Type Culture Collection (ATCC) and maintained according


to the manufacturer’s instructions. CD14+ monocytes were purifiedfrom the peripheral blood mononuclear cells of healthy donors usingCD14 MicroBeads (Miltenyi Biotec) according to the manufacturer’sinstructions, as previously described (50). To generate MDMs, isolatedCD14+monocyteswere plated on a 10-cmpetri dish at 10 × 106 cells perdish. Monocytes were stimulated with macrophage colony-stimulatingfactor (25 ng/ml) (R&D Systems) in macrophage serum-free medium(Thermo Fisher Scientific) for 7 days. MDMs were then maintained inDulbecco’s modified Eagle’s medium (Thermo Fisher Scientific)containing 10% fetal bovine serum (FBS) (HyClone Laboratories),25 mM Hepes (Quality Biology), and gentamicin (5 mg/ml) (ThermoFisher Scientific) before they were used in experiments. HSV-2 was ob-tained from Advanced Biotechnologies Inc., and viral titers weredetermined by plaque-forming assays with Vero cells (ATCC) (51).Antibodies used in this study were as follows: anti-STING, anti-MyD88, anti–b-catenin, anti-Ku80, anti-IRF1, anti-IRF7, and anti-Flagantibodies were from Cell Signaling Technology; anti-Ku70 and anti–DNA-PKcs antibodies were from Abcam; and anti-IRF3 antibody wasfrom OriGene. Alexa Fluor 488– and Alexa Fluor 555–labeledsecondary antibodies were purchased from Cell Signaling Technology.

Gel electrophoresis and Western blotting analysisCell lysates were prepared with radioimmunoprecipitation assay buffer(Boston BioProducts) in the presence of protease inhibitor cocktail(Sigma-Aldrich) andHalt phosphatase inhibitor cocktail (Thermo FisherScientific). Nuclear protein was extracted with the Nuclear Extract Kit(Active Motif) according to the manufacturer’s instructions. The proteinconcentrations of the cell lysates were quantified with the bicinchoninicacid protein assay (Thermo Fisher Scientific) to ensure that equalamounts of total protein were loaded in each well of NuPAGE 4 to12% bis-tris gels (Thermo Fisher Scientific). Proteins were transferredonto a nitrocellulose membrane and analyzed byWestern blotting withthe appropriate antibodies, which was followed by incubation withhorseradish peroxidase–conjugated secondary antibodies and detectionof bands with ECL plus Western blotting detection reagents (GEHealthcare). Band intensities were analyzed with National Institutesof Health Image J software (http://rsbweb.nih.gov/ij/).

PlasmidsThe PCR2.1 plasmid (Thermo Fisher Scientific) was digested with EcoRI, and this digested plasmid was used as a noncoding DNA stimulant.The Myc-tagged STING expression vector was constructed as follows.STING-encoding complementary DNA (cDNA) was synthesized fromthe total cellular RNA of HEK 293 cells using the Superscript First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific). Thereverse-transcribed cDNA encoding STING was subcloned intopEF6/Myc-His (ThermoFisher Scientific). The insertionwas confirmedby DNA sequencing with BigDye version 3.0 (Applied Biosystems).Other expression vectors used in this studywere constructedwith a sim-ilar method. The cDNA encoding Ku70 was cloned into pEF1-Flag(Thermo Fisher Scientific). The DNA encoding the full-length IFNL1promoter region [993 base pairs (bp)] was amplified from HEK 293genomic DNA with the Expand high-fidelity PCR system (RocheMolecular Biochemical) with a primer pair (5′-GAGCTCAAAC-CAATGGCAGAAGCTCC-3′ and 5′-AGATCTTGGCTAAATCG-CAACTGCTTCC-3′). The 993-bp fragment was subcloned andinserted into the vector pGL4.10 (Promega). The presence of theintended fragment without any unexpected mutations was confirmedby DNA sequencing, and this vector was named pGL4–IFN-l1. The

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plasmid pTK-RL, which expresses Renilla luciferase under the controlof HSV thymidine kinase promoter, was obtained from Promega andused to normalize experimental variations.

IFN-l1 reporter assay293T cells (30 × 103 cells per well) were seeded in six-well plates andcotransfected with 100 ng of pGL4–IFN-l1, 10 ng of pTK-RL, and theappropriate amounts of STING expression vector, wild-type Ku70 ex-pression vector, or mutant Ku70 expression vector. The cells were thentransfected on day 2 with 1 mg of linearized DNA (as a stimulant), andthe cells were then collected for the luciferase assay. The luciferase assaywas performedwithDual-Glo luciferase assay system reagents (Promega).Relative luciferase activity was calculated on the basis of the ratio of theluminescence of firefly luciferase to that of Renilla luciferase.

RNA extraction and real-time RT-PCRTotal cellular RNAwas isolated from cells with the RNeasy isolation kit(Qiagen). The cDNA was synthesized from total RNA with TaqManreverse transcription reagents (Thermo Fisher Scientific) with randomhexamers as primers, according to themanufacturer’s instructions. Therelative abundance of IFNL1mRNAwasmeasured by quantitative RT-PCR on a CFX96 real-time system (Bio-Rad); a two-temperature cycleof 95°C for 15 s and 60°C for 1 min (repeated for 40 cycles) was used.Relative quantities of IFNL1 transcripts were calculated with the DDCtmethod with GAPDHmRNA as a reference. Normalized samples wereexpressed relative to the averageDCt value for controls to obtain relativefold changes in mRNA abundance.

Generation of STING KO cellsSTING KO cells were generated from HEK 293 cells with the CRISPR/Cas9 genomic editing kit (OriGene) according to the manufacturer’sinstructions. HEK 293 cells (5 × 105) were transfected with 600 ng ofhCas9 target guide RNA and 600 ng of donor vector in a six-well tissueculture plate. The cells were split at a 1:10 dilution 2 days after transfec-tion. After an additional 3 days in culture, the cells were split again at a1:10 dilution. This procedure was repeated seven times. The cells werethen transferred to 10-cm dishes and cultured withmedium containingpuromycin (2 mg/ml) (Thermo Fisher Scientific). The culture mediumwas changed every 2 to 3 days. Individual cell colonies were isolated bylimiting dilution. After 1 to 2 weeks, the cells were observed under themicroscope, and cells from those wells containing only one cell colonywere selected and allowed to expand from a 96-well to a 6-well plate.

Coimmunoprecipitation assaysHEK 293T cells (1 × 106) were seeded onto a 100-mm dish and trans-fected with a Flag-tagged Ku70/Ku70 mutant expression vector and aMyc-tagged STING expression vector using a TransIT-293 Transfec-tion Kit (Mirus Bio LLC). The cells were further transfected on day 2with 10 mg of linearized DNA (as a stimulant). The cells were lysed inPierce IP lysis buffer (Thermo Fisher Scientific) with 10% glycerol(Sigma-Aldrich). The lysates were centrifuged at 10,000g for 10 minat 4°C to remove cell debris. The supernatants were immunoprecipi-tated with an anti-Flag agarose (Sigma-Aldrich) or anti–c-Myc agarose(Thermo Fisher Scientific) and then analyzed byWestern blotting withanti-FLAG or anti-STING antibodies.

Confocal microscopyCells grown on 35-mm glass bottom dishes were fixed in 4% para-formaldehyde, blocked in blocking buffer [1× phosphate-buffered sa-


line, 5% normal goat serum (Cell Signaling), and 0.1% saponin (AlfaAesar)] for 1 hour, stained overnight with primary antibodies (dilutedat 1:200) at 4°C, and then incubated with Alexa Fluor 488– or AlexaFluor 555–labeled secondary antibodies (1:1000) for 1 hour. Bottom-coated coverslips were mounted with ProLong Diamond AntifadeMountant withDAPI (Thermo Fisher Scientific). Imageswere capturedon an LSM 710 scanning confocal microscope.

Lentivirus packaging and transductionA lentiviral plasmid containing an expression cassette encodingSTING-specific shRNA was purchased from OriGene. Lentiviral parti-cles were packaged in 293T cells according to the manufacturer’s in-structions. Harvested lentivirus-containing cell culture medium wasconcentrated with a lenti concentrator (OriGene). The lentiviral parti-cleswere titrated on the basis of their abundance of theHIVp24 antigenmeasured with an HIV-1 p24 ELISA kit (PerkinElmer). For lentiviraltransductions, human macrophages were seeded on six-well plates to70% confluence and inoculated with lentiviruses at an MOI of 50in culture medium containing 2% FBS in the presence of polybrene(8 mg/ml). Twenty-four hours after the cells were transduced, the me-dium was replaced by fresh complete culture medium, and the cellswere then incubated for a further 72 hours to enable protein knockdownto occur.

Enzyme-linked immunosorbent assayThe amounts of IFN-a, IFN-b, IFN-l1, and IFN-l2/3 proteins in cell cul-ture medium were measured with the VeriKine-HS Human IFN-a AllSubtype ELISA Kit (PBL Assay Science), the VeriKine-HS HumanIFN-b Serum ELISA Kit (PBL Assay Science), the human IL-29/IFN-l1DuoSet ELISA (R&D Systems), and the DIY Human IFN-l2/3 (IL-28A/B)ELISA (PBL Assay Science) kits, respectively, according to the manufac-turers’ protocols. The minimum detectable concentrations of IFN-a,IFN-b, IFN-l1, andIFN-l2/3were1.95,1.2,62.5, and125pg/ml, respectively.

Statistical analysisResults were representative of at least three independent experiments.The values are expressed as means ± SD of individual samples. Statis-tical significance was determined by Student’s t test. P < 0.05 wasconsidered to indicate a statistically significant difference between theexperimental groups.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/488/eaah5054/DC1Fig. S1. Time course of theDNA-stimulated inductionof IFN-a, IFN-b, IFN-l1, and IFN-l2/3 production.Fig. S2. Comparison of the endogenous amounts of b-catenin and MyD88 proteins betweenHEK 293 cells and 293T cells.Fig. S3. Analysis of the production of IFN-a, IFN-b, and IFN-l2/3 in THP-1 cells transfected with DNA.Fig. S4. Knockdown of IFI16 has no effect on the Ku70-mediated induction of IFN-l1production in response to exogenous DNA.Fig. S5. Ku80 and DNA-PKcs are not involved in the Ku70-mediated induction of IFN-l1production in response to exogenous DNA.Fig. S6. The activation of IRF1, IRF3, and IRF7 involves the Ku70-mediated induction of IFNproduction in response to exogenous DNA.Fig. S7. Knockdown of STING inhibits the production of IFN-a, IFN-b, and IFN-l2/3 by humanMDMs transfected with DNA or infected with HSV-2.

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Acknowledgments: We thank H. Young for discussion, S. Lockett and K. Peifley for technicalsupport on the confocal microscope, and Q. Chen for assistance with supplies. Funding:This project was funded in whole or in part with federal funds from the National Cancer Institute,NIH under contract no. HHSN261200800001E. The content of this publication does not necessarilyreflect the reviews or policies of the U.S. Department of Health and Human Services nor doesmention of trade names, commercial products, or organizations imply endorsement by theU.S. government. Author contributions: H.S. designed and performed experiments; H.S., H.C.L.,and T.I. discussed and interpreted the data; T.I. and H.I. supported constructing the Ku70 mutants;X.J. and B.T.S. analyzed the confocal images; M.Z. performed mass spectrometry analysis; andH.S., H.C.L., and T.I. wrote the manuscript. Competing interests: The authors declare that theyhave no competing interests.

Submitted 6 July 2016Resubmitted 27 September 2016Accepted 30 June 2017Published 18 July 201710.1126/scisignal.aah5054

Citation: H. Sui, M. Zhou, H. Imamichi, X. Jiao, B. T. Sherman, H. C. Lane, T. Imamichi, STING isan essential mediator of the Ku70-mediated production of IFN-l1 in response to exogenousDNA. Sci. Signal. 10, eaah5054 (2017).

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exogenous DNA1 in response toλSTING is an essential mediator of the Ku70-mediated production of IFN-

Hongyan Sui, Ming Zhou, Hiromi Imamichi, Xiaoli Jiao, Brad T. Sherman, H. Clifford Lane and Tomozumi Imamichi

DOI: 10.1126/scisignal.aah5054 (488), eaah5054.10Sci. Signal.

phase of the antiviral IFN response to DNA viruses.type III IFN production was slower than that of type I IFN. Together, these data suggest a role for STING in the late mediator of type I IFN production. However, in the Ku70 pathway, IRF1 and IRF7 were required in addition to IRF3, andKu70 translocated from the nucleus to the cytosol to interact with the adaptor protein STING, a well-characterized

1 in human cells exposed to cytosolic DNA or infected with the DNA virus HSV-2.λproduction of the type III IFN IFN-inducedinvestigated the mechanism by which the DNA protein kinase (DNA-PK) component Ku70, a DNA repair protein,

et al.transcription factor IFN regulatory factor 3 (IRF3) to induce expression of genes encoding type I IFNs. Sui part of the antiviral response. Various cytosolic DNA sensors and their downstream effectors stimulate activation of the

When cells sense cytosolic DNA, such as occurs during viral infection, they produce type I interferons (IFNs) as1λSTINGing viruses with IFN-

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STING is an essential mediator of the Ku70-mediated production … · 293T cells (Fig. 3C). We next investigated whether the removal of STING from HEK 293 cellswould modifythe DNA-stimulated