1. Volume 20, Issue 3, Pages 329-341 (September 2016)
STING Requires the Adaptor TRIF to Trigger Innate Immune Responses to Microbial Infection 
Xin Wang, Tanmay Majumdar, Patricia Kessler, Evgeny Ozhegov, Ying Zhang, Saurabh Chattopadhyay, Sailen Barik, Ganes C. Sen 
Cell Host & Microbe 
Volume 20, Issue 3, Pages (September 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 20, 329-341DOI: (10.1016/j.chom.2016.08.002)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1 Gene Induction by STING Requires TRIF
(A) TRIF−/− or TRIF+ (TRIF expression restored in TRIF−/− cells) MEFs were treated with the STING-ligands c-di-GMP, cGAMP, or the vehicle, DMSO for 4 hr and IFN-β mRNA levels were measured by qRT-PCR.
(B) WT and TRIF−/− MEFs were treated with c-di-GMP for 6 hr and the levels of TNF-α and IFN-β secreted to the culture medium were quantitated by ELISA.
(C) WT and TRIFlps2/lps2 (a different TRIF−/− line) MEFs were treated with c-di-GMP for the indicated lengths of time and the induction of Ifit2 protein was monitored by western blot analysis.
(D) Ifit2 induction in WT and TRIFlps2/lps2 bone marrow-derived macrophages after 6 hr of c-di-GMP treatment was analyzed by western blot.
(E) Human IFIT1 induction was analyzed in WT or TRIF−/− HeLa-M cells (generated by the CRISPR-CAS9 technique) after 6 hr of cGAMP treatment by western blot.
(F) Human IFN-β mRNA induction was analyzed in TRIF−/− and TRIF+ HT1080 (TRIF expression restored in TRIF−/− cells) after 6 hr of cGAMP treatment.
(G) Microarray analysis was done with RNAs extracted from TRIF−/− or TRIF+ cells after 4 hr of treatment with c-di-GMP or DMSO; results for the top 15 most highly induced mRNAs are shown (in A, B, and F, mean ± SEM, n = 3).
See also Figure S1 and Table S1.
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 2 TRIF-Interaction Is Required for STING Signaling
(A) WT or TRIF−/− MEFs were treated with c-di-GMP for the indicated time. The phosphorylated and total proteins (IRF3, TBK1, and Akt) were analyzed by western blot.
(B) WT or TRIF−/− MEFs were treated as in (A). The phosphorylated and total IκB-α were analyzed by western blot.
(C) WT and TRIF−/− MEFs were treated with c-di-GMP (+) or DMSO (−) for 4 hr, when cell lysates were immunoprecipitated with anti-STING antibody. The immunoprecipitates and the cell lysates were analyzed by western blot for the indicated proteins.
(D) Silver staining analysis of TRIF purified by Ni-NTA chromatography from HEK293T cells.
(E) In vitro interaction of STING and TRIF: cell lysate from HEK293T-expressing HA-STING was incubated with anti-HA antibody (HA) or normal IgG (IgG). The STING-bound complex was affinity purified by Protein A/G agarose and the beads were washed with high salt buffer to remove the associated proteins. Purified TRIF (TRIF) or equal amount of BSA (BSA) was mixed with 10-fold of carrier BSA and incubated with bead-bound STING/IgG in the presence of 150 mM NaCl. The proteins on beads were submitted to western blot.
(F) TRIF, STING, the cytosolic protein α-tubulin, and the ER protein calnexin were analyzed by western blot. 0.9% of total cell lysate (T), 0.9% of cytoplasmic fraction (C), and 6% of ER fraction of untreated MEF were used for the analysis. All samples were electrophoresed on the same gel, but not in adjacent lanes.
See also Figure S2.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 3 The C Terminus of TRIF Is Required for STING Interaction
(A) Left: schematic diagram of mouse TRIF and its mutants; right: mouse HA-STING was co-expressed with FL or various deletion mutants of mouse His/Xpress-TRIF in HEK293T cells. From the lysates, His-tagged TRIF was pulled down (PD) by Ni-NTA and the levels of STING and TRIF bound to the beads were measured by western blot (IB).
(B) Similar analyses, as in (A), with human HA-STING and His/V5-TRIF.
(C) TRIF−/− MEFs were transfected with FL or truncated mouse TRIF mutants. After 16 hr of transfection, the cells were treated with c-di-GMP for 4 hr and IFN-α mRNA and IL6 mRNA level were measured by qRT-PCR (mean ± SEM, n = 3).
See also Figure S3.
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 4 STING C Terminus Domain Interacts with TRIF C Terminus Domain
(A) Left: schematic diagram of STING and its mutants; right: V5-TRIF was expressed by itself (vector) or co-expressed with FL or deletion mutants of HA-STING in HEK293T cells, the cell lysates were immunoprecipitated with anti-HA antibody and the immunoprecipitates were analyzed for TRIF and STING.
(B) V5-TRIF was co-expressed with WT or mutant HA-STING in HEK293T cells. TRIF was immunoprecipitated with anti-V5 or normal IgG and STING and TRIF levels in the precipitates were analyzed by western blot.
(C) HA-STING CTD (220–378 amino acid, aa) was co-expressed with His-V5 doubly tagged TRIF mutants (ΔC, 1–541 or CTD, 542–732); STING/TRIF interaction was analyzed by Ni-NTA pull down of TRIF followed by western blot with HA or V5 antibodies.
See also Figure S3.
Cell Host & Microbe  , DOI: ( /j.chom )
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7. Figure 5 TRIF Promotes STING Dimerization and Mobilization
(A) WT or TRIF−/− MEFs cells were transfected with HA-STING and treated with c-di-GMP for 4 hr; STING was immunoprecipitated with anti-HA and the immunoprecipitates were analyzed by western blot. Dimeric STING appears as a 74 kDa band.
(B) Myc-STING was co-transfected with HA-STING (+) or empty vector (−) to WT and TRIF−/− MEFs, as indicated. After 4 hr of c-di-GMP treatment, HA-STING/Myc-STING interaction was measured by anti-HA immunoprecipitation and western blot with anti-Myc or anti-HA.
(C) WT MEFs were transfected with TRIF (+) or an empty vector (−). STING dimerization was analyzed by immunoprecipitation with anti-STING antibody followed by western blot with the same antibody.
(D) TRIF−/− MEFs were transfected with HA-STING combined with empty vector (Vector), His/Xpress-TRIF 385–732 (ΔN), or His/Xpress-TRIF full length (TRIF). STING dimerization was analyzed by immunoprecipitation with anti-HA antibody followed by western blot.
(E) WT and TRIF−/− MEFs were transfected with GFP-STING and treated with c-di-GMP or DMSO for 2 hr. The cells were fixed and analyzed by confocal microscopy. GFP-STING distribution in a representative cell from every group is shown; only the cell in the upper left showed punctate signals, others had diffuse signals.
(F) From (E), the percentages of cells with punctate or diffuse signals in GFP-STING positive cells were calculated; the numbers of cells counted are indicated in each column.
(G) TRIF−/− MEFs were transfected with WT or mutant (V155M) HA-STING. STING dimerization was analyzed by immunoprecipitation with anti-HA antibody followed by western blot.
(H) TRIF−/− MEFs were transfected with empty vector (Vector), WT, or V155M STING. Various parameters of STING signaling (as indicated) were analyzed, 16 hr after transfection, by western blot.
See also Figure S4.
Cell Host & Microbe  , DOI: ( /j.chom )
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8. Figure 6 TRIF Restricts HSV-1 Replication by Promoting STING Signaling
(A) Different MEFs, as indicated, were infected with HSV-1 at 5 moi. Ifit2 induction was monitored by western blot at indicated time post infection.
(B) WT and TRIF−/− MEFs were infected with HSV-1 for 4 hr and induction of the indicated mRNAs was analyzed by qRT-PCR.
(C) HSV-1 DNA levels in infected MEFs were quantitated by qPCR at the indicated time post virus adsorption.
(D) MEFs expressing the WT or mutant (P625L) TRIF were treated with c-di-GMP for 4 hr and gene induction was quantitated by qRT-PCR.
(E) HEK293T cells were transfected with HA-STING alone (NTC) or in combination with empty vector (EV), WT Xpress-TRIF, or P625L Xpress-TRIF, as indicated. STING was immunoprecipitated from the lysates with HA antibody and STING dimerization was analyzed by western blot with anti-HA. TRIF and actin levels in the lysates are shown at the bottom.
(F) HEK293T cells were transfected with HA-STING in combination with empty vector (vector), WT His/Xpress-TRIF, or P625L His/Xpress-TRIF (as indicated). TRIF was pulled down from the lysates by Ni-NTA and bound STING and TRIF were measured by western blot. The levels of transfected STING and TRIF in the lysates are shown at the bottom.
(G) At 16 hr post infection, HSV-1 DNA levels in TRIF−/− MEFs expressing WT or mutant TRIF were analyzed by qPCR (in B–D and G, mean ± SEM, n = 3).
See also Figure S5.
Cell Host & Microbe  , DOI: ( /j.chom )
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9. Figure 7 TRIF Restricts HSV-1 Replication and Pathogenesis in Mouse
(A) Kinetics of survival of WT and TRIF−/− infected mice (p < by log rank test).
(B) Kinetics of weight changes.
(C) Virus loads in mouse brains (mean ± SEM, n = 3).
Cell Host & Microbe  , DOI: ( /j.chom )
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