1. Volume 64, Issue 1, Pages 105-119 (October 2016)
TRIM14 Inhibits cGAS Degradation Mediated by Selective Autophagy Receptor p62 to Promote Innate Immune Responses 
Meixin Chen, Qingcai Meng, Yunfei Qin, Puping Liang, Peng Tan, Lian He, Yubin Zhou, Yongjun Chen, Junjiu Huang, Rong-Fu Wang, Jun Cui 
Molecular Cell 
Volume 64, Issue 1, Pages (October 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2016 64, 105-119DOI: (10.1016/j.molcel.2016.08.025)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
TRIM14 Is a Positive Regulator of cGAS-Induced Type I IFN Signaling
(A) Luciferase activity in HEK293T cells transfected with 32 TRIMs along with Flag-cGAS, Flag-STING, and an IFN-β luciferase reporter (IFN-β-luc). The dotted line represents the average. EV, empty vector. Gray point, negative control. Red point (numeral 14), TRIM14 with cGAS and STING. Dark points, other TRIMs with cGAS and STING.
(B) Luciferase activity in HEK293T cells transfected with luciferase reporters for ISRE (ISRE-luc), IFN-β (IFN-β-luc), or NF-κB (NF-κB-luc), together with EV or increasing amount of HA-TRIM14 (wedge).
(C and D) Luciferase activity in wild-type (WT), MAVS knockout (KO) (C) or NEMO KO (D) HEK293T cells transfected with Flag-cGAS, Flag-STING, Flag-RIG-I (N), HA-TRIM14, or EV along with IFN-β-luc.
(E) ELISA of IFN-β production in the supernatants of THP-1 cells treated with indicated TRIM14-specific (siTRIM14) or scrambled (Scr) siRNA, followed by HSV-1 infection for 24 hr. The TRIM14 protein was analyzed by immunoblot.
(F) Immunoassay of THP-1 cells transfected with TRIM14-specific or scrambled siRNA, followed by HSV-1 infection for the indicated time points.
(G) Quantitative PCR (Q-PCR) analysis of THP-1 cells transfected with indicated TRIM14-specific or scrambled siRNA, followed by HSV-1 infection for the indicated time points.
Data in (A)–(E) and (G) are expressed as means ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
TRIM14 Deficiency Impairs Resistance to HSV-1 Infection by Producing Less IFN-β In Vivo
(A) Immunoassay of WT or Trim14−/− BMDMs, pMs, or MEFs by HSV-1 infection for the indicated time periods.
(B) ELISA of IFN-β production in the supernatants of WT or Trim14−/− BMDMs by HSV-1 or VSV infection for 24 hr.
(C and D) Q-PCR analysis of WT and Trim14−/− BMDMs infected with HSV-1 (C) or VSV (D) for the indicated time periods.
(E) Q-PCR analysis of IFNB mRNA in WT or Trim14−/− BMDMs reconstituted with vector or TRIM14 and infected with HSV-1 for the indicated time points.
(F and G) Q-PCR analysis of HSV-1 or VSV genome in BMDMs (F) or MEFs (G) treated as in (C).
(H) Determination of HSV-1 loads in liver by TCID50 assay 24 hr after WT and Trim14−/− mice (n = 3 per group) were intravenously injected with HSV-1 (3 × 108 pfu per g).
(I) Q-PCR analysis of HSV-1 genome of WT (n = 6) and Trim14−/− (n = 6) mice given intravenous injection of HSV-1 (3 × 108 pfu per g).
(J) Survival of 7-week-old WT and Trim14−/− mice given intravenous injection of HSV-1 (n = 6). p < 0.05.
(K) Pathology of WT and Trim14−/− mice in response to HSV-1. Scale bar, 100 μm. H&E staining of lung sections from mice in (H).
(L) Q-PCR analysis of IFN-β expression in liver from WT and Trim14−/− mice in (H).
(M) ELISA of IFNβ, TNF-α, and IL-6 production in sera from WT and Trim14−/− mice in (H).
Data in B–J, L, and M are expressed as means ± SD of at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 TRIM14 Associates With and Stabilizes cGAS
(A–C) Coimmunoprecipitation and immunoassay of extracts of HeLa cells (A), THP-1 cells (B), or PBMCs (C) infected for the indicated time points with HSV-1. WCL, whole-cell lysates.
(D and E) Confocal microscopy of HeLa cells (D) or BMDMs (E) infected with HSV-1 for 24 hr. Scale bar, 10 μm.
(F) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with HA-TRIM14 together with Flag-cGAS (FL), Flag-cGAS (DBD), or Flag-cGAS (NTase+CTD).
(G) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-cGAS together with Myc-TRIM14 (FL), Myc-TRIM14 (ΔB-Box), Myc-TRIM14 (ΔB-Box-CC), Myc-TRIM14 (ΔPRYSPRY), or Myc-TRIM14 (PRYSPRY).
(H) Immunoassay of extracts of HEK293T cells transfected with Flag-cGAS and increasing doses of HA-TRIM14 (wedge). Below, RT-PCR analysis of cGAS mRNA; GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase) serves as a loading control.
(I) Immunoassay of cGAS, MAVS, STING proteins in lysates of WT, and Trim14−/− BMDMs or pMs infected for the indicated time points with HSV-1.
(J) Immunoassay of HEK293T cells transfected with Flag-cGAS (FL), Flag-cGAS (DBD), or Flag-cGAS (NTase+CTD) along with or without HA-TRIM14.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 TRIM14 Blocks Autophagic Degradation of cGAS
(A) Immunoassay of extracts of WT or Beclin1 KO (BECN 1 KO) HEK293T cells transfected with Myc-cGAS and treated with Earle’s balanced salt solution (EBSS) for the indicated time points.
(B) Immunoassay of extracts of WT or ATG5 KO A549 cells infected with HSV-1 for the indicated time points.
(C) Immunoassay of extracts of HEK293T cells transfected with Flag-cGAS (FL), Flag-cGAS (DBD), and Flag-cGAS (NTase + CTD) and treated with or without EBSS.
(D) Immunoassay of extracts of TRIM14-inducible HeLa cells treated with increasing doses of doxycycline (Dox) for 24 hr (wedge) and treated with or without bafilomycin A1 (Baf, 0.2 μM) for 8 hr (left) or MG132 (10 μM) for 6 hr (right). ddH2O or dimethyl sulfoxide (DMSO) was used for controls of Baf and MG132, respectively.
(E) Immunoassay of extracts of WT and ATG5 KO HEK293T cells transfected with Flag-cGAS and HA-TRIM14.
(F) Immunoassay of extracts of WT or TRIM14 KO THP-1 cells treated with or without Baf along with or without HSV-1 infection.
(G) Immunoassay of extracts of WT or Trim14−/− MEFs treated with or without EBSS for 2 hr or rapamycin (Rapa, 100 nM) for 24 hr.
(H) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with various combinations of expression vector for Flag-cGAS, GFP-LC3, and HA-TRIM14.
(I) Coimmunoprecipitation and immunoassay of extracts of WT or TRIM14 KO HeLa cells infected with or without HSV-1 for 24 hr.
(J) Confocal microscopy of TRIM14-inducible HeLa cells treated with EBSS and Dox (50 ng/ml) or not. Scale bar, 10 μm. Statistics shown refer to the puncta formation by cGAS-LC3 or LC3 in the indicated samples.
Data in (J) are presented in mean ± SEM. ∗∗∗p < See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
TRIM14 Inhibits p62-Mediated Selective Autophagic Degradation of cGAS
(A) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, OPTN, Tollip, or NBR1, together with Myc-cGAS.
(B and C) Immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-cGAS, HA-p62, and treated with EBSS for 0, 30, or 60 min (B) or with Rapa for 24 hr (C).
(D) Immunoprecipitation and immunoassay of extracts of TRIM14-inducible HeLa cells treated with Dox (50 ng/ml) or not.
(E) Confocal microscopy of TRIM14-inducible HeLa cells treated with EBSS and Dox (50 ng/ml) or not. Scale bar, 10 μm. Statistics of the puncta formation by cGAS-p62 or p62 are determined in the indicated samples.
(F) Immunoprecipitation and immunoassay of extracts of WT and Trim14−/− BMDMs with or without HSV-1 infection.
(G) Confocal microscopy of WT and Trim14−/− BMDMs treated with Baf along with EBSS. Scale bar, 10 μm.
Data in (E) are presented in mean ± SEM. ∗∗∗p < See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 TRIM14 Impairs the K48-Linked Ubiquitination of cGAS
(A) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with various combinations of plasmid for cGAS and p62 constructs.
(B) Coimmunoprecipitation and immunoassay of extracts of HeLa cells treated with EBSS along with or without Baf.
(C) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with various combinations of plasmid for Flag-cGAS, HA-Ub, HA-K48-Ub, or HA-K48R-Ub, then treated with HT-DNA (2 μg/ml) or infected with HSV-1.
(D) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-cGAS and indicated cGAS mutants along with HA-K48-Ub, then treated with Baf and EBSS.
(E) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-cGAS, Flag-cGAS (K414R), and HA-p62.
(F) Immunoassay of extracts of HEK293T cells transfected with various combinations of plasmid for Flag-cGAS, HA-TRIM14, and HA-K48-Ub and treated with EBSS along with Chloroquine phosphate (CQ, 20 μM), followed by two-step immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA.
(G) Coimmunoprecipitation and immunoassay of extracts of WT or TRIM14 KO HeLa cells transfected with EV, Myc-TRIM14, or Myc-TRIM14-ΔPRYSPRY and infected with or without HSV-1.
(H) Confocal microscopy of WT and Trim14−/− BMDMs cells treated with Baf along with EBSS or not.
(I) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with various combinations of plasmid for Flag-cGAS, Flag-cGAS (K414R), HA-K48-Ub, and HA-TRIM14, then treated with Baf and EBSS.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
TRIM14 Recruits USP14 to Cleave the K48-Linked Ubiquitination of cGAS
(A) Immunoassay of extracts of HEK293T cells transfected with various combinations of plasmid for Flag-cGAS, HA-TRIM14, Flag-BRCC3, Myc-USP14, and Myc-USP14 (C114A).
(B) Coimmunoprecipitation and immunoassay of extracts of WT and TRIM14 KO HeLa cells transfected with various combinations of plasmid for Flag-cGAS, Myc-USP14, Myc-USP(CA), and HA-K48-Ub constructs.
(C) Coimmunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-USP14 together with Myc-TRIM14 (FL), Myc-TRIM14 (ΔB-Box), Myc-TRIM14 (ΔB-Box-CC), Myc-TRIM14 (ΔPRYSPRY), or Myc-TRIM14 (PRYSPRY).
(D) Immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-cGAS, HA-TRIM14 and HA-USP14.
(E) Coimmunoprecipitation and immunoassay of extracts of WT or TRIM14 KO HeLa cells transfected with EV, Myc-TRIM14, or Myc-TRIM14-ΔPRYSPRY and infected with or without HSV-1.
(F) Confocal microscopy of WT or TRIM14 KO HeLa cells stimulated with or without HSV-1. Scale bar, 10 μm.
(G) Immunoprecipitation and immunoassay of extracts of WT or USP14 KO THP-1 cells infected with or without HSV-1 for 24 hr.
(H) Luciferase activity of WT or USP14 KO HEK293T cells transfected with an IFN-β luciferase reporter, together with various combinations of plasmid for EV, Flag-cGAS, Flag-STING, HA-TRIM14, Myc-USP14, and Myc-USP14 (C114A).
(I) Schematic representation of cGAS-mediated type I interferon signaling regulated by TRIM14/USP14 axis.
See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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