Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a, b ) Susceptibility of Dgcr8 -/- , Dicer -/- and parental cells to TMEV infection after inhibition of IRF3 (BX795) ( a ) and Nf-κB (BMS345541) ( b ), normalized to mock-treated cells. ( c ) Heat map of significantly differentially expressed proteins (p<0.05) in the absence ( Dgcr8 -/- ) or presence ( Dgcr8 resc ) of miRNAs identified by STRING analysis. ( d ) Western blot analysis of MAVS expression in miRNA-deficient cells ( Dgcr8 -/- and Dicer -/- , lanes 2 and 5), wild-type counterparts ( Dgcr8 +/+ and Dicer +/+ , lanes 1 and 4) and respective rescued ESCs lines ( Dgcr8 resc and Dicer resc , lanes 3 and 6). MAVS quantification normalized to Tubulin and relative to wild-type levels is shown at the top of the panel. 10.7554/eLife.44171.010 Figure 3—source data 1. Source data of mass spectrometry results.

Article Snippet: Cells were pre-incubated with the inhibitors BX795, which blocks the phosphorylation of the kinases TBK1 and IKKε, and consequently IRF3 activation and IFN-β production (10 μM, Synkinase) and the inhibitor BMS345541, which targets IKβα, IKKα and IKKβ and consequently NF-κβ signalling (10 μM, Cayman Chemical) for 45 min before infection with TMEV.

Techniques: Infection, Inhibition, Western Blot, Expressing, Mass Spectrometry

Journal: Immunity

Article Title: Unanchored K48-linked poly-ubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase mediated antiviral response

doi: 10.1016/j.immuni.2014.04.018

Figure Lengend Snippet: (A) Schematics of the IFN mediated IKKε-dependent and IKKε-independent signaling for ISG induction. (B) WT, Ikbke-/- or Ddx58-/- MEFs were transfected with ISG54 luciferase reporter plasmid together with empty vector or TRIM6 plasmid. Twenty-four h p.t, cells were stimulated with IFNβ (100 U/ml) for 16 h, followed by luciferase assay. Data shown are representative of three independent experiments and depicted is the mean ± SD (n=3). (C,D,F,G,H) A549 cells were tranfected with TRIM6 siRNAs followed by stimulation with IFNβ (100U/ml). Cells were harvested for (C) RT-qPCR analysis of ISG mRNA, (D) IB or (F) EMSA analysis. (E) hDCs transduced with lentiviruses expressing TRIM6 shRNAs were stimulated with IFNβ (100U/ml) and subjected to IB. (G-H) A549 cells were transfected with TRIM6 siRNAs for 40 h and subsequently pre-treated with IFNβ (100U/ml) for 16 h. Cells were then infected with IAV expressing GFP (PR8-GFP) for 16 h. Cells were visualized by fluorescence microscopy (G) and quantified by FACS (H). *p < 0.05; **p < 0.01; ***p < 0.001, N.S. (not significant). See also Figure S4.

Article Snippet: We thank Christopher F. Basler for kindly providing the IKKε deletion plasmids.

Techniques: Transfection, Luciferase, Plasmid Preparation, Quantitative RT-PCR, Transduction, Expressing, Infection, Fluorescence, Microscopy

Journal: Immunity

Article Title: Unanchored K48-linked poly-ubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase mediated antiviral response

doi: 10.1016/j.immuni.2014.04.018

Figure Lengend Snippet: (A) HEK-293T cells were transfected with FLAG-IKKε together with HA-Ub WT (lanes 1-2, 4-6, 15) or different Ub mutants, and GST-TRIM6 (lane 6-14) or GST-RBCC (TRIM6 lacking SPRY domain: lane 15), or (B) FLAG-IKKε with HA-TRIM6 or TRIM6 C15A RING mutant. IP was performed with anti-FLAG beads. (C) Luciferase assay of HEK-293T cells transfected with ISG54 reporter together with TRIM6 WT or C15A mutant, and treated with IFNβ (100 U/ml) or (D) tranfected with TRIM6 siRNA followed by transfection with ISG54 reporter and TRIM6 plasmids and IFNβ treatment (100U/ml) 24 hr later. Depicted is the mean ± SD (n = 3). (E) hDCs transduced with lentiviruses expressing TRIM6 shRNAs were stimulated with IFNβ (100U/ml) and subjected to IKKε IP. (F) BALB/c mice were treated i.n. with 12,000 units of universal IFN. Lungs were collected and IKKε IP was performed. (G-H) Silencing of TRIM6 in vivo. BALB/c mice were treated i.n. with TRIM6-targeting or non-targeting PPMO at 24 and 48 hr prior to i.n. infection with 100 pfu IAV PR8. Lungs were collected for virus titer (G), depicted is the mean ± SD; n=3, or at 48 hrs pi. for IKKε IP (H). See also Figure S5.

Article Snippet: We thank Christopher F. Basler for kindly providing the IKKε deletion plasmids.

Techniques: Transfection, Mutagenesis, Luciferase, Transduction, Expressing, In Vivo, Infection, Virus

Journal: Immunity

Article Title: Unanchored K48-linked poly-ubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase mediated antiviral response

doi: 10.1016/j.immuni.2014.04.018

Figure Lengend Snippet: (A) HEK-293T cells, transfected with FLAG-IKKε plasmid together with empty vector or HA-TRIM6 and His-tagged Ub, were subjected to His-pulldown under denaturing conditions. (B) HEK-293T cells were transfected with empty vector or FLAG-IKKε together with HA-Ub, and GST-TRIM6 in the presence or absence of the isopeptidase T (IsoT) which cleaves only unanchored Ub. At 30 h p.t, IP with anti-FLAG antibody. FLAG-JAK1 was used as a control of covalently bound Ub which IsoT cannot cleave (right side). (C) ISG54 reporter assay of HEK-293T cells transfected with IKKε and TRIM6 in the presence or absence of IsoT, or (D) in the presence or absence of IsoT, OTU domain of the CCHFV or a mutant (C40A and H151A) lacking catalytic activity, followed by IFNβ treatment. Data shown is representative of 3 independent experiments (mean ± SD, n=3). (E-F) IKKε is recruited to “TRIM6 Ub-rich bodies”. (E) Confocal microscopy of HeLa cells transfected with HA-TRIM6 and FLAG-Ub, or (F) with HA-TRIM6, FLAG-IKKε and His-Ub. See also Figure S6.

Article Snippet: We thank Christopher F. Basler for kindly providing the IKKε deletion plasmids.

Techniques: Transfection, Plasmid Preparation, Control, Reporter Assay, Mutagenesis, Activity Assay, Confocal Microscopy