human ifn beta promoter  (Addgene inc)

Journal: PLoS ONE

Article Title: RO 90-7501 Enhances TLR3 and RLR Agonist Induced Antiviral Response

doi: 10.1371/journal.pone.0042583

Figure Lengend Snippet: (A) Structure of RO 90–7501. (B) 293TLR3/IFNβLuc and 293TLR3/NFκBLuc cells were mock treated or treated with 10 µM of RO 90–7501, 2 µg/ml of poly I:C, alone or in combination for 16 h. Luciferase activities were normalized to mock-treated controls and expressed as fold of induction (mean +/− standard deviation, n = 8). P values were calculated using the two-tailed student t test. (C) 293TLR3/IFNβLuc and 293TLR3/NFκBLuc cells were mock treated or treated with 2 µg/ml of poly I:C in the absence or presence of the indicated concentrations of RO 90–7501 for 16 h. The effects of RO 90–7501 on the IFN-β promoter and NFκB reporter activation were normalized to mock-treated controls and expressed as fold of induction of luciferase activity (mean +/− standard derivation, n = 3). P values were calculated using the two-tailed student t test, with * indicates P<0.05 compared to poly I:C treated alone. (D) 293TLR3/IFNβLuc cells were mock treated or treated with indicated concentrations of RO 90–7501 for 16 h. Cell viability was determined by a MTT assay and the results were plotted as the dose-dependent absorbance values at 570 nm.

Article Snippet: 293TLR3HA cells were co-transfected with plasmids expressing firefly luciferase under the control of a human IFN-β promoter (pIFNβ-Luc) or an artificial promoter containing four repetitive consensus NFκB binding sites (Clontech) and pcDNA3 (Invitrogen) at a molar ratio of 10 to 1.

Techniques: Luciferase, Standard Deviation, Two Tailed Test, Activation Assay, Activity Assay, MTT Assay

Journal: PLoS ONE

Article Title: RO 90-7501 Enhances TLR3 and RLR Agonist Induced Antiviral Response

doi: 10.1371/journal.pone.0042583

Figure Lengend Snippet: (A) 293TLR3HA cells were mock treated or treated with 10 µM of RO 90–7501, 2 µg/ml poly I:C, alone or in combination. The cells were harvested at the indicated time since start of the treatment. The levels of IFN-β and β-actin mRNAs were determined by semi-quantitative RT-PCR. (B) THP-1 cells were mock treated or treated with the indicated concentrations of poly I:C in the absence or presence of 10 µM RO 90–7501 for 6 h. The levels of secreted type I IFN were then determined with HEK-Blue IFN-α/β cells using culture media transferred from treated THP-1 cells. The level of ISG54 promoter driven SEAP expression in the supernatant was then determined with QUANTI-Blue and expressed as absorbance values at 655 nm, normalized to mock treated controls (mean +/− standard derivations, n = 8). P values were calculated using the two-tailed student t test. (C) 293TLR3HA cells were mock treated or treated with the indicated concentrations of poly I:C in the absence or presence of 10 µM RO 90–7501 for 6 h. Secreted IL-8 in culture media from treated 293TLR3HA cells were determined with a human IL-8 ELISA kit, and expressed as absorbance values at 450 nm (mean +/− standard derivations, n = 3).

Article Snippet: 293TLR3HA cells were co-transfected with plasmids expressing firefly luciferase under the control of a human IFN-β promoter (pIFNβ-Luc) or an artificial promoter containing four repetitive consensus NFκB binding sites (Clontech) and pcDNA3 (Invitrogen) at a molar ratio of 10 to 1.

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: USP27X negatively regulates antiviral signaling by deubiquitinating RIG-I

doi: 10.1371/journal.ppat.1008293

Figure Lengend Snippet: (A–C) HEK293T cells were co-transfected with the indicated expression plasmids and luciferase reporter constructs driven by promoters of IFNβ (A), NF-κB (B), or ISRE (C), as well as Renilla as an internal control. Twenty-four hours after transfection, cells were infected with SeV for 12 h. Cell lysates were analyzed for luciferase assays (upper panel) and immunoblotting assays (lower panels). (D–F) HEK293T cells were transfected with plasmids expressing USP27X or empty vector. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times, and abundance of mRNAs encoding IFNB1 (D), TNFα (E) and IFIT1 (F) was measured by qRT-PCR. (G) HEK293T cells were transfected with plasmids expressing USP27X or empty vector. Twenty-four hours after transfection, the cells were infected with SeV for 9 h. Cell lysates were resolved by native gel electrophoresis (upper panel) or SDS-PAGE (lower panels) and analyzed with the indicated antibodies. (H) HEK293T cells were transfected with plasmids expressing USP27X or empty vector. Twenty-four hours after transfection, the cells were infected with SeV for the indicated times, followed by immunoblotting. The data shown in (A–F) are from one representative experiment of at least three independent experiments [mean ± SD of duplicate experiments in (A–C) or triplicate experiments in (D–F)]. The two-tailed Student’s t-test was used to analyze statistical significance. ** P < 0.01; *** P < 0.001 versus control groups.

Article Snippet: A genome-wide siRNA screen using the Silencer Human Ubiquitin siRNA Library (Dharmacon) was conducted by transfection a HEK293T stable reporter cell line that expresses firefly luciferase driven by a human IFNβ promoter.

Techniques: Transfection, Expressing, Luciferase, Construct, Control, Infection, Western Blot, Plasmid Preparation, Quantitative RT-PCR, Nucleic Acid Electrophoresis, SDS Page, Two Tailed Test

Journal: PLoS Pathogens

Article Title: USP27X negatively regulates antiviral signaling by deubiquitinating RIG-I

doi: 10.1371/journal.ppat.1008293

Figure Lengend Snippet: (A) HEK293T cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, the cells were lysed for Co-IP with anti-Flag agarose beads, followed by immunoblotting. The expression levels of transfected proteins in whole cell lysates (WCL) are shown in the bottom panels. (B) HepG2 cells were transfected with Flag-USP27X, or Flag-USP27X-72 expression vector or empty vector. Twenty-four hours after transfection, the cells were mock-infected or infected with SeV for 18 h. Cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting with the indicated antibodies. (C) HEK293T cells were transfected with Myc-USP27X expression vector or empty vector. Twenty-four hours after transfection, the cells were mock-infected or infected with SeV for 12 h. Cell lysates were immunoprecipitated with anti-RIG-I antibody, followed by immunoblotting. (D) HEK293T cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cells were mock-infected or infected with SeV (50HA) for 9 h. The cells were fixed, stained with the anti-Flag (red) and anti-Myc (green) antibodies, and observed by confocal microscopy. (E–G) HEK293T cells were co-transfected with the indicated expression plasmids along with luciferase reporter constructs driven by promoters of IFNβ (E), ISRE (F) or NF-κB (G) as well as Renilla as an internal control. Twenty-four hours after transfection, the cells were lysed for luciferase assays (upper panel) and immunoblotting assays (lower panels). (H–J) Similar to (E–G), except that expression plasmids MAVS were used instead of RIG-I(N). The data shown in (E–J) are from one representative experiment of at least three independent experiments (mean ± SD of duplicate experiments). The two-tailed Student’s t-test was used to analyze statistical significance. **P < 0.01; n.s. not significant versus control groups.

Article Snippet: A genome-wide siRNA screen using the Silencer Human Ubiquitin siRNA Library (Dharmacon) was conducted by transfection a HEK293T stable reporter cell line that expresses firefly luciferase driven by a human IFNβ promoter.

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Infection, Immunoprecipitation, Staining, Confocal Microscopy, Luciferase, Construct, Control, Two Tailed Test

Journal: PLoS Pathogens

Article Title: USP27X negatively regulates antiviral signaling by deubiquitinating RIG-I

doi: 10.1371/journal.ppat.1008293

Figure Lengend Snippet: (A) HEK293T cells were transfected with the indicated expression plasmids. Cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting. (B) Similar to (A) except that HEK293T cells were transfected with different expression plasmids as indicated. (C–E) HEK293T cells were co-transfected with the indicated expression plasmids along with luciferase reporter constructs driven by promoters of IFNβ (C), ISRE (D) or NF-κB (E). Twenty-four hours after transfection, the cells were infected with SeV for 12 h. The cells were lysed for luciferase assays (upper panel) and immunoblotting assays (lower panels). (F) HEK293T cells were co-transfected with the indicated expression plasmids and IFNβ reporter. Twenty-four hours after transfection, the cells were lysed for luciferase assays (upper panel) and immunoblotting assays (lower panels). (G) HEK293T cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, the cells were infected with SeV for 9 h. The cells were lysed for measurement of IFNB1 mRNA levels by qRT-PCR. The data shown in C–G are from one representative experiment of at least three independent experiments [mean ± SD of duplicate experiments in (C–F) or triplicate experiments in (G)]. The two-tailed Student’s t-test was used to analyze statistical significance. * P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant versus control groups.

Article Snippet: A genome-wide siRNA screen using the Silencer Human Ubiquitin siRNA Library (Dharmacon) was conducted by transfection a HEK293T stable reporter cell line that expresses firefly luciferase driven by a human IFNβ promoter.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Luciferase, Construct, Infection, Quantitative RT-PCR, Two Tailed Test, Control

Journal: PLoS Pathogens

Article Title: USP27X negatively regulates antiviral signaling by deubiquitinating RIG-I

doi: 10.1371/journal.ppat.1008293

Figure Lengend Snippet: (A–C) HEK293T cells were co-transfected with the indicated expression plasmids, and luciferase reporter constructs driven by promoters of IFNβ (A), ISRE (B) or NF-κB (C). Twenty-four hours after transfection, the cells were lysed for luciferase assays (upper panel) and immunoblotting assays (lower panels). (D) HEK293T cells were transfected with the indicated expression plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting. (E–G) HEK293T cells were transfected with USP27X and MDA5(N) together with HA-tagged wild-type Ub (HA-Ub) (E), HA-Ub-K63 (F), or HA-Ub-K48 (G) plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag beads, followed by immunoblotting analysis with the indicated antibodies. The expression levels of transfected proteins in whole cell lysates (WCL) are shown in the bottom panels. The data shown in A–C are from one representative experiment of at least three independent experiments (mean ± SD of duplicate experiments). The two-tailed Student’s t-test was used to analyze statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001 versus control groups.

Article Snippet: A genome-wide siRNA screen using the Silencer Human Ubiquitin siRNA Library (Dharmacon) was conducted by transfection a HEK293T stable reporter cell line that expresses firefly luciferase driven by a human IFNβ promoter.

Techniques: Transfection, Expressing, Luciferase, Construct, Western Blot, Immunoprecipitation, Two Tailed Test, Control