high molecular weight polyic  (Takeda)

Journal: bioRxiv

Article Title: DDX50 is a viral restriction factor that enhances TRIF-dependent IRF3 activation

doi: 10.1101/2020.08.06.239376

Figure Lengend Snippet: Firefly luciferase activity of WT or Ddx50 −/− MEFs transfected with plasmids encoding Firefly Luciferase under the Ifnβ promoter and Renilla. (A) Cells were left untreated or treated with 5 μg/ml extracellular PolyIC (epIC), transfected with 5 μg/ml PolyIC (pIC) for 6 h or infected with Sendai virus (SeV) for 24 h. (B) WT or Ddx50 −/− MEFs were transfected with lipofectamine only or 5 μg/ml pIC for 7 h, and the fold induction of Isg56 , Cxcl10 or Ifnb mRNA levels, relative to Gapdh, were analysed by RT-qPCR. (C) Secreted levels of CXCL10 and IL-6 in the medium at 7 h post transfection with PolyIC or (D) 4.5 h post infection with SeV were analysed by ELISA. (E) Firefly Luciferase activity of WT or Ddx50 −/− HEK293Ts transfected with plasmids encoding Firefly Luciferase under the Ifnβ promoter and Renilla. Cells were infected for 24 h with SeV or left untreated. Data are representative of at least three independent experiments. (F) Secreted levels of CXCL10 in the medium at 24 h post infection of WT or Ddx50 −/− HEK293Ts with SeV, were analysed by ELISA. Data are representative of at least three independent experiments. Statistical significance shown for WT stimulated vs KO stimulated.

Article Snippet: Twenty-four h post-transfection, cells were stimulated with IL-1α (Invivogen) or TNFα (Invivogen) at 100 ng/ml or transfected with 5 μg/ml high molecular weight (HMW) PolyIC (Invivogen) using Liopfectamine 2000 (Invitrogen), or mock-transfected with lipofectamine only, or treated exogenously with 5 μg/ml PolyIC, or left unstimulated for 6 h in DMEM or MEM with 2 % FBS.

Techniques: Luciferase, Activity Assay, Transfection, Infection, Virus, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: DDX50 is a viral restriction factor that enhances TRIF-dependent IRF3 activation

doi: 10.1101/2020.08.06.239376

Figure Lengend Snippet: (A-C) WT MEFs transduced with pLDT-EV and Ddx50 −/− MEFs transduced with pLDT-EV or pLDT- Ddx50 were transfected with lipofectamine only or 5 μg/ml PolyIC for 7 h. (A) Isg56 , Cxcl10 or Ifnb mRNA levels, relative to Gapdh, were analysed by RT-qPCR and (B) secreted CXCL10 was measured by ELISA. Representative of at least two independent experiments. Statistical significance shown for WT stimulated vs KO stimulated. (C) Expression of DDX50 was confirmed by SDS-PAGE and immunoblotting.

Article Snippet: Twenty-four h post-transfection, cells were stimulated with IL-1α (Invivogen) or TNFα (Invivogen) at 100 ng/ml or transfected with 5 μg/ml high molecular weight (HMW) PolyIC (Invivogen) using Liopfectamine 2000 (Invitrogen), or mock-transfected with lipofectamine only, or treated exogenously with 5 μg/ml PolyIC, or left unstimulated for 6 h in DMEM or MEM with 2 % FBS.

Techniques: Transduction, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, SDS Page, Western Blot

Journal: bioRxiv

Article Title: DDX50 is a viral restriction factor that enhances TRIF-dependent IRF3 activation

doi: 10.1101/2020.08.06.239376

Figure Lengend Snippet: (A and B) Representative immunoblot of phosphorylated IRF3 at (A) Ser396 or (B) Ser386 (pIRF3) for (A) WT or Ddx50 −/− MEFs transfected with lipofectamine only or 5 μg/ml PolyIC for 3 and 6 h or (B) WT and DDX50 −/− HEK293Ts untreated or infected with SeV for 18 h. Level of IRF3 phosphorylation was calculated by densitometry, relative to α-tubulin (A) or actin (B) and is representative of at least two independent experiments. (C) Representative immunoblot following transfection of WT MEFs with 2.5 μg/ml PolyIC for the indicated times and isolation of the cytoplasmic (cyt) and nuclear fractions (nuc). Immunoblots were stained for DDX50 or α-tubulin and laminA/C as cytoplasmic and nuclear fraction controls, respectively. Representative of three independent experiments. (D) Immunofluorescence staining for DDX50 localisation. HeLa cells were transfected with pLDT-DDX50-HA and left uninfected (NI) or infected for 1.5 h with SeV. DDX50 localisation was visualised using an anti-HA antibody. DAPI was used to stain the nucleus. Representative of three independent experiments. ( E ) Luciferase activity of WT or Ddx50 −/− MEFs co-transfected with EV or indicated plasmids along with plasmids encoding Firefly Luciferase under the Ifnβ promoter and Renilla as an internal control. Experiments shown are representative of at least three independent experiments. Statistical significance shown for WT stimulated vs KO stimulated.

Article Snippet: Twenty-four h post-transfection, cells were stimulated with IL-1α (Invivogen) or TNFα (Invivogen) at 100 ng/ml or transfected with 5 μg/ml high molecular weight (HMW) PolyIC (Invivogen) using Liopfectamine 2000 (Invitrogen), or mock-transfected with lipofectamine only, or treated exogenously with 5 μg/ml PolyIC, or left unstimulated for 6 h in DMEM or MEM with 2 % FBS.

Techniques: Western Blot, Transfection, Infection, Isolation, Staining, Immunofluorescence, Luciferase, Activity Assay