Journal: Nucleic Acids Research

Article Title: siRNA enhances DNA-mediated interferon lambda-1 response through crosstalk between RIG-I and IFI16 signalling pathway

doi: 10.1093/nar/gkt844

Figure Lengend Snippet: STING, TBK1 and IRF3 activation are involved in siRNA-induced IFN-λ1 production. ( A ) 293T cells were co-transfected with pGL4-IFN-λ1 and pRL-TK, together with expression vectors of RIG-I, IFI16, STING and TBK1 as indicated combinations. The cells in the condition of C1–C5 were transfected with an empty vector to ensure each well has the same amounts of DNA transfection as the condition C6. Cells were then stimulated with siRNA and plasmid DNA transfection on day 2 and day 3, respectively. On day 4, cells were collected for luciferase assay. Relative luciferase activity was determined by the ratio of firefly to Renilla readings. ( B ) HeLa cells were transfected with or without 10 nM Non-human Ctrl siRNA along with two different siRNA targeting IRF3 (siIRF3-1 and siIRF3-2). Cells were then treated with IFN-α (1000 U/ml) followed by DNA transfection on day 2 and day 3, respectively. RNA was extracted 6 h after DNA transfection. Relative IFN-λ1 mRNA expression level was measured by real-time RT-PCR and compared with the cells treated with IFN-α and DNA transfection but without siRNA transfection. ** P < 0.001 (Student’s t -test). ( C ) Top: HeLa cells were stimulated with 10 nM siRNA and then treated with IFN-α (1000 U/ml) and plasmid DNA transfection (1 µg). Cells were lysed at different time points after DNA transfection. HeLa cells stimulated by Poly(I:C) for 1 h were set as a positive control for IRF3 activation. The collected cell lysates were separated on native PAGE and analysed by western blot using anti-IRF3 antibody. Bottom: band intensity of IRF3 dimer relative to that of IRF3 monomer was assessed by NIH Image J.

Article Snippet: Antibodies used in this study were as follows: anti-RIG-I (Cell Signalling Technology, Danvers, MA, USA); anti-IFI16 (Santa Cruz, Santa Cruz, CA, USA); anti-STING (LifeSpan BioSciences, Seattle, WA, USA); anti-TBK1 and anti-Flag (Cell Signalling Technology) and anti-IRF3 (Santa Cruz).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Quantitative RT-PCR, Positive Control, Clear Native PAGE, Western Blot

Journal: Nucleic Acids Research

Article Title: siRNA enhances DNA-mediated interferon lambda-1 response through crosstalk between RIG-I and IFI16 signalling pathway

doi: 10.1093/nar/gkt844

Figure Lengend Snippet: siRNA also enhances HSV-1–mediated IFN-λ1 production. ( A ) HeLa cells were transfected with or without 10 nM Non-human Ctrl siRNA along with siRNA targeting RIG-I (siRIG-I-1 and siRIG-I-2), IFI16 (siIFI16-1 and siIFI16-2), TBK1 (siTBK1-1 and siTBK1-2) or STING (siSTING-1 and siSTING-2). Cells were then treated with IFN-α (1000 U/ml) followed by HSV-1 infection at a MOI of 5. RNA was extracted 6 h after virus infection. Relative IFN-λ1 mRNA expression level was measured by real-time RT-PCR and compared with the cells treated with IFN-α and HSV-1 infection (no siRNA in the figure, the IFN-λ1 induction under this condition has ∼20-folds increase compared with that of uninfected cells). ( B ) Whole-cell lysates were collected, and an equal amount of total proteins were separated using SDS-PAGE and subjected to western blot to detect RIG-I, IFI16, TBK1 and STING expression. β-actin was used as a loading control. ( C ) Impact of the overexpression of RIG-I, IFI16, TBK1 and STING on the siRNA-enhanced HSV-1–mediated IFN-λ1 production. HeLa cells were transfected with RIG-I, IFI16, STING or TBK1 expression vector and followed by a transfection of Non-human Ctrl siRNA. An empty vector was transfected to the cells without RIG-I, IFI16, STING or TBK1 overexpression to ensure each condition with the same amount of DNA transfection. The transfected cells were then treated with IFN-α (100 U/ml) for 6 h (a minor IFN-α treatment to confer a lower IFN-λ1 induction) followed by HSV-1 infection at a MOI of 5. RNA was extracted 6 h after HSV-1 infection. Relative IFN-λ1 mRNA expression level was measured by real-time RT-PCR and compared with the cells without siRNA transfection. ( D ) Whole-cell lysates of the cells were collected, and an equal amount of total proteins was loaded on SDS-PAGE and then subjected to western blot to detect RIG-I, IFI16, TBK1 and STING.

Article Snippet: Antibodies used in this study were as follows: anti-RIG-I (Cell Signalling Technology, Danvers, MA, USA); anti-IFI16 (Santa Cruz, Santa Cruz, CA, USA); anti-STING (LifeSpan BioSciences, Seattle, WA, USA); anti-TBK1 and anti-Flag (Cell Signalling Technology) and anti-IRF3 (Santa Cruz).

Techniques: Transfection, Infection, Expressing, Quantitative RT-PCR, SDS Page, Western Blot, Over Expression, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: siRNA enhances DNA-mediated interferon lambda-1 response through crosstalk between RIG-I and IFI16 signalling pathway

doi: 10.1093/nar/gkt844

Figure Lengend Snippet: A simplified view of siRNA-enhanced DNA-mediated IFN-λ1 signalling pathway. ( A ) RIG-I and IFI16 formed a protein complex in the presence of siRNA. ( B ) IFI16 dissociated with the RIG-I–siRNA–IFI16 complex and bound with invaded DNA or DNA virus. ( C ) The dissociation of IFI16 initiated the downstream STING-TBK1-IRF3 signalling pathway. RIG-I/MAVS pathway also partially contributed the type III IFN induction pathway, but how MAVS interact with downstream TBK1 or STING remains unclear, where is indicated by a question mark and a dashed line.

Article Snippet: Antibodies used in this study were as follows: anti-RIG-I (Cell Signalling Technology, Danvers, MA, USA); anti-IFI16 (Santa Cruz, Santa Cruz, CA, USA); anti-STING (LifeSpan BioSciences, Seattle, WA, USA); anti-TBK1 and anti-Flag (Cell Signalling Technology) and anti-IRF3 (Santa Cruz).

Techniques: