mouse genome-scale sirna libraries Search Results


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    Millipore large scale shrna lentivirus pool transduction
    SeV infection induces CTNNB1 stabilization and nuclear translocation. (A) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, CTNNB1 phosphorylated at Ser552, NFKBIA (IκBα) phosphorylated at Ser32, IFIT1, DDX58 and SeV protein HN of HEK <t>293T</t> transduced with lentivirus-expressing <t>shRNA</t> 45 targeting CTNNB1 and shRNA NT (control) for four days and subjected to SeV infection for 0, 4, 8, 24, 32 or 48 hours. (B–D) Fold induction of IFNB1 (B), IFIT1 (C) and TNF (D) mRNA levels in HEK 293T cells treated as described in (A). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (E) Confocal analysis of HEK 293T cells using Hoechst, anti-CTNNB1 active form and anti-IRF3 antibodies without virus infection or following 16 hours infection with SeV. Nuclear detection of IRF3 and CTNNB1 is identified by white arrows in infected cells.
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    SeV infection induces CTNNB1 stabilization and nuclear translocation. (A) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, CTNNB1 phosphorylated at Ser552, NFKBIA (IκBα) phosphorylated at Ser32, IFIT1, DDX58 and SeV protein HN of HEK <t>293T</t> transduced with lentivirus-expressing <t>shRNA</t> 45 targeting CTNNB1 and shRNA NT (control) for four days and subjected to SeV infection for 0, 4, 8, 24, 32 or 48 hours. (B–D) Fold induction of IFNB1 (B), IFIT1 (C) and TNF (D) mRNA levels in HEK 293T cells treated as described in (A). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (E) Confocal analysis of HEK 293T cells using Hoechst, anti-CTNNB1 active form and anti-IRF3 antibodies without virus infection or following 16 hours infection with SeV. Nuclear detection of IRF3 and CTNNB1 is identified by white arrows in infected cells.
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    Image Search Results


    SeV infection induces CTNNB1 stabilization and nuclear translocation. (A) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, CTNNB1 phosphorylated at Ser552, NFKBIA (IκBα) phosphorylated at Ser32, IFIT1, DDX58 and SeV protein HN of HEK 293T transduced with lentivirus-expressing shRNA 45 targeting CTNNB1 and shRNA NT (control) for four days and subjected to SeV infection for 0, 4, 8, 24, 32 or 48 hours. (B–D) Fold induction of IFNB1 (B), IFIT1 (C) and TNF (D) mRNA levels in HEK 293T cells treated as described in (A). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (E) Confocal analysis of HEK 293T cells using Hoechst, anti-CTNNB1 active form and anti-IRF3 antibodies without virus infection or following 16 hours infection with SeV. Nuclear detection of IRF3 and CTNNB1 is identified by white arrows in infected cells.

    Journal: PLoS Pathogens

    Article Title: Genome-wide RNAi Screen Reveals a New Role of a WNT/CTNNB1 Signaling Pathway as Negative Regulator of Virus-induced Innate Immune Responses

    doi: 10.1371/journal.ppat.1003416

    Figure Lengend Snippet: SeV infection induces CTNNB1 stabilization and nuclear translocation. (A) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, CTNNB1 phosphorylated at Ser552, NFKBIA (IκBα) phosphorylated at Ser32, IFIT1, DDX58 and SeV protein HN of HEK 293T transduced with lentivirus-expressing shRNA 45 targeting CTNNB1 and shRNA NT (control) for four days and subjected to SeV infection for 0, 4, 8, 24, 32 or 48 hours. (B–D) Fold induction of IFNB1 (B), IFIT1 (C) and TNF (D) mRNA levels in HEK 293T cells treated as described in (A). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (E) Confocal analysis of HEK 293T cells using Hoechst, anti-CTNNB1 active form and anti-IRF3 antibodies without virus infection or following 16 hours infection with SeV. Nuclear detection of IRF3 and CTNNB1 is identified by white arrows in infected cells.

    Article Snippet: Large-scale lentiviral shRNA production 293T cells were transfected using PEI with 6 µg pLKO.1-puro encoding shRNA targeting WNT2B (TRCN0000033376 and TRCN0000033377), WNT9B (TRCN0000062069 and TRCN0000062070), WLS (TRCN0000133858 and TRCN0000133999), CTNNB1 (TRCN0000003843, TRCN0000003844 and TRCN0000003845) or shRNA non-target (NT, Sigma), 1.5 µg pMDLg/pRRE, 1.5 µg pRSV-REV and 3 µg pVSVg as previously described .

    Techniques: Infection, Translocation Assay, Transduction, Expressing, shRNA, Quantitative RT-PCR

    Pharmacological inhibition of GSK3 stabilizes an active CTNNB1 pool and negatively regulates antiviral innate immunity. (A–B) Relative fold induction of IFNB1 -driven reporter activity (A) and fold induction of CTNNB1-TCF/LEF dependent reporter activity TOP-flash (B) in HEK 293T cells transduced with lentivirus-expressing shRNA NT (control) or shRNA 45 targeting CTNNB1 for four days and subjected to GSK3 inhibition with BIO (5 µM) or BIO-acetoxime (10 µM) and SeV infection for 16 hours. (C) Immunoblot analysis of autophosphorylated GSK3α/β at Tyr279/216, dephosphorylated active CTNNB1 at Ser37/Thr41 and IFIT1 in HEK 293T cells treated with GSK3 inhibitors BIO (5 µM) or BIO-acetoxime (10 µM) and infected with SeV for 16 hours. (D) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, IFIT1 and phosphorylated NFKBIA (IκBα) at Ser32 in SeV-infected HEK 293T cells treated as described in (A–B) and treated with BIO (5 µM) or BIO-acetoxime (10 µM). (E) Relative fold induction of TOP-flash, IFNB1 , ISG56 , NF-κB and EF1α -driven reporter activity in HEK 293T cells subjected to GSK3 inhibition with BIO (5 µM), TCF/LEF interaction inhibition with PNU74654 (6 µM) or both inhibitors prior to SeV infection for 16 hours.

    Journal: PLoS Pathogens

    Article Title: Genome-wide RNAi Screen Reveals a New Role of a WNT/CTNNB1 Signaling Pathway as Negative Regulator of Virus-induced Innate Immune Responses

    doi: 10.1371/journal.ppat.1003416

    Figure Lengend Snippet: Pharmacological inhibition of GSK3 stabilizes an active CTNNB1 pool and negatively regulates antiviral innate immunity. (A–B) Relative fold induction of IFNB1 -driven reporter activity (A) and fold induction of CTNNB1-TCF/LEF dependent reporter activity TOP-flash (B) in HEK 293T cells transduced with lentivirus-expressing shRNA NT (control) or shRNA 45 targeting CTNNB1 for four days and subjected to GSK3 inhibition with BIO (5 µM) or BIO-acetoxime (10 µM) and SeV infection for 16 hours. (C) Immunoblot analysis of autophosphorylated GSK3α/β at Tyr279/216, dephosphorylated active CTNNB1 at Ser37/Thr41 and IFIT1 in HEK 293T cells treated with GSK3 inhibitors BIO (5 µM) or BIO-acetoxime (10 µM) and infected with SeV for 16 hours. (D) Immunoblot analysis of dephosphorylated active CTNNB1 at Ser37/Thr41, IFIT1 and phosphorylated NFKBIA (IκBα) at Ser32 in SeV-infected HEK 293T cells treated as described in (A–B) and treated with BIO (5 µM) or BIO-acetoxime (10 µM). (E) Relative fold induction of TOP-flash, IFNB1 , ISG56 , NF-κB and EF1α -driven reporter activity in HEK 293T cells subjected to GSK3 inhibition with BIO (5 µM), TCF/LEF interaction inhibition with PNU74654 (6 µM) or both inhibitors prior to SeV infection for 16 hours.

    Article Snippet: Large-scale lentiviral shRNA production 293T cells were transfected using PEI with 6 µg pLKO.1-puro encoding shRNA targeting WNT2B (TRCN0000033376 and TRCN0000033377), WNT9B (TRCN0000062069 and TRCN0000062070), WLS (TRCN0000133858 and TRCN0000133999), CTNNB1 (TRCN0000003843, TRCN0000003844 and TRCN0000003845) or shRNA non-target (NT, Sigma), 1.5 µg pMDLg/pRRE, 1.5 µg pRSV-REV and 3 µg pVSVg as previously described .

    Techniques: Inhibition, Activity Assay, Transduction, Expressing, shRNA, Infection

    CTNNB1 is a negative regulator of antiviral innate immunity. (A) IFNB1 promoter-driven luciferase activity in HEK 293T cells transduced with lentivirus-expressing shRNA NT (control) or 3 shRNAs targeting CTNNB1 for four days and subjected to SeV infection for 16 hours. (B) Immunoblot analysis of CTNNB1 and IFIT1 in HEK 293T cells transduced with 3 lentivirus-expressing shRNAs targeting CTNNB1 and infected with SeV. (C) Immunoblot analysis of CTNNB1, IFIT1 and DDX58 in SeV-infected HEK 293T cells previously transduced with a shRNA or transfected with a pool of four siRNAs targeting CTNNB1 for three days. (D–F) Fold induction of IFNB1 (D), DDX58 (E) and TNF (F) mRNA levels in HEK 293T cells treated as described in (C). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (G) ELISA quantification of secreted IFN-β protein in supernatant of HEK 293T cells treated as described in (A). (H–I) Fold induction of IFNB1 promoter-driven luciferase activity (H) and of CTNNB1-TCF/LEF dependent reporter activity (TOP-flash, I) following dose-dependent transfection of CTNNB1-expressing plasmid (50, 100, 150 and 200 ng) for 48 hours in SeV-infected HEK 293T cells.

    Journal: PLoS Pathogens

    Article Title: Genome-wide RNAi Screen Reveals a New Role of a WNT/CTNNB1 Signaling Pathway as Negative Regulator of Virus-induced Innate Immune Responses

    doi: 10.1371/journal.ppat.1003416

    Figure Lengend Snippet: CTNNB1 is a negative regulator of antiviral innate immunity. (A) IFNB1 promoter-driven luciferase activity in HEK 293T cells transduced with lentivirus-expressing shRNA NT (control) or 3 shRNAs targeting CTNNB1 for four days and subjected to SeV infection for 16 hours. (B) Immunoblot analysis of CTNNB1 and IFIT1 in HEK 293T cells transduced with 3 lentivirus-expressing shRNAs targeting CTNNB1 and infected with SeV. (C) Immunoblot analysis of CTNNB1, IFIT1 and DDX58 in SeV-infected HEK 293T cells previously transduced with a shRNA or transfected with a pool of four siRNAs targeting CTNNB1 for three days. (D–F) Fold induction of IFNB1 (D), DDX58 (E) and TNF (F) mRNA levels in HEK 293T cells treated as described in (C). qRT-PCR determination represents the average mRNA RQ normalized versus ACTIN and HPRT1 mRNA. (G) ELISA quantification of secreted IFN-β protein in supernatant of HEK 293T cells treated as described in (A). (H–I) Fold induction of IFNB1 promoter-driven luciferase activity (H) and of CTNNB1-TCF/LEF dependent reporter activity (TOP-flash, I) following dose-dependent transfection of CTNNB1-expressing plasmid (50, 100, 150 and 200 ng) for 48 hours in SeV-infected HEK 293T cells.

    Article Snippet: Large-scale lentiviral shRNA production 293T cells were transfected using PEI with 6 µg pLKO.1-puro encoding shRNA targeting WNT2B (TRCN0000033376 and TRCN0000033377), WNT9B (TRCN0000062069 and TRCN0000062070), WLS (TRCN0000133858 and TRCN0000133999), CTNNB1 (TRCN0000003843, TRCN0000003844 and TRCN0000003845) or shRNA non-target (NT, Sigma), 1.5 µg pMDLg/pRRE, 1.5 µg pRSV-REV and 3 µg pVSVg as previously described .

    Techniques: Luciferase, Activity Assay, Transduction, Expressing, shRNA, Infection, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    Genome-wide gene silencing study of virus-induced innate immune responses and bioinformatics analyses. (A) Schematic representation of the primary genome-wide screen and secondary screens. HEK 293T cells stably expressing the luciferase gene under the control of the IFNB1 promoter were transduced with arrayed lentiviruses combining three shRNAs per gene (primary screen) or with five individual shRNA-expressing lentiviruses for each gene hit (secondary screens) in a 96-well format. After 72 hours, cells were challenged with SeV virus (primary and confirmation screens) or transfected with polyinosinic∶polycytidylic acid (polyI∶C), MAVS- or IRF3(5D)-expressing plasmids (secondary screens) for 16 hours before measuring IFNB1 promoter-driven luciferase activity. (B) Decision tree of primary screen and summary data of gene hits obtained in secondary and validation screens. Selected gene hits (114) that were confirmed and validated with endogenous IFNB1 screens by qRT-PCR induced a modulation of more than 25% of the IFNB1 promoter activity with at least two independent shRNAs following SeV infection. Prioritized gene hits (59) for which knockdown of the target gene was greater than 40% with two independent shRNAs are also identified. (C) Schematic representation of confirmation and secondary assays for epistasis analysis of gene hits acting on the signaling cascade leading to IFNB1 production. SeV infection (primary and confirmation screens), polyI∶C (dsRNA mimetic), MAVS or IRF3(5D) expressing plasmids transfection (secondary screens) were used to activate innate immune response. A non-specific assay was used to discard gene hits affecting nonimmune-related transcription by measuring transcriptional activity of EF1α constitutive promoter. (D) Heat map indicating modulation of IFNB1 promoter activity following silencing of control genes in confirmation and secondary assays (log 2 scale). The functional profiling data for controls and gene hits allow classification within four functional groups: I - SeV specific, II - cytoplasmic dsRNA sensing, III - MAVS-dependent signaling, IV - nuclear import or transcription factor-dependent process. (E) Functional profiling data of 114 gene hits confirmed with at least two shRNAs in the SeV confirmation screen and further validated using endogenous IFNB1 mRNA quantification by qRT-PCR. Manual clustering was performed to classify each gene hit in one of the four functional groups. (F) qRT-PCR validation data of the endogenous IFNB1 mRNA levels and target gene knockdown efficiency in transduced cells with each lentivirus-expressing shRNA for the 114 gene hits. (G) Enriched Gene Ontology (GO) biological process and molecular function terms (P

    Journal: PLoS Pathogens

    Article Title: Genome-wide RNAi Screen Reveals a New Role of a WNT/CTNNB1 Signaling Pathway as Negative Regulator of Virus-induced Innate Immune Responses

    doi: 10.1371/journal.ppat.1003416

    Figure Lengend Snippet: Genome-wide gene silencing study of virus-induced innate immune responses and bioinformatics analyses. (A) Schematic representation of the primary genome-wide screen and secondary screens. HEK 293T cells stably expressing the luciferase gene under the control of the IFNB1 promoter were transduced with arrayed lentiviruses combining three shRNAs per gene (primary screen) or with five individual shRNA-expressing lentiviruses for each gene hit (secondary screens) in a 96-well format. After 72 hours, cells were challenged with SeV virus (primary and confirmation screens) or transfected with polyinosinic∶polycytidylic acid (polyI∶C), MAVS- or IRF3(5D)-expressing plasmids (secondary screens) for 16 hours before measuring IFNB1 promoter-driven luciferase activity. (B) Decision tree of primary screen and summary data of gene hits obtained in secondary and validation screens. Selected gene hits (114) that were confirmed and validated with endogenous IFNB1 screens by qRT-PCR induced a modulation of more than 25% of the IFNB1 promoter activity with at least two independent shRNAs following SeV infection. Prioritized gene hits (59) for which knockdown of the target gene was greater than 40% with two independent shRNAs are also identified. (C) Schematic representation of confirmation and secondary assays for epistasis analysis of gene hits acting on the signaling cascade leading to IFNB1 production. SeV infection (primary and confirmation screens), polyI∶C (dsRNA mimetic), MAVS or IRF3(5D) expressing plasmids transfection (secondary screens) were used to activate innate immune response. A non-specific assay was used to discard gene hits affecting nonimmune-related transcription by measuring transcriptional activity of EF1α constitutive promoter. (D) Heat map indicating modulation of IFNB1 promoter activity following silencing of control genes in confirmation and secondary assays (log 2 scale). The functional profiling data for controls and gene hits allow classification within four functional groups: I - SeV specific, II - cytoplasmic dsRNA sensing, III - MAVS-dependent signaling, IV - nuclear import or transcription factor-dependent process. (E) Functional profiling data of 114 gene hits confirmed with at least two shRNAs in the SeV confirmation screen and further validated using endogenous IFNB1 mRNA quantification by qRT-PCR. Manual clustering was performed to classify each gene hit in one of the four functional groups. (F) qRT-PCR validation data of the endogenous IFNB1 mRNA levels and target gene knockdown efficiency in transduced cells with each lentivirus-expressing shRNA for the 114 gene hits. (G) Enriched Gene Ontology (GO) biological process and molecular function terms (P

    Article Snippet: Large-scale lentiviral shRNA production 293T cells were transfected using PEI with 6 µg pLKO.1-puro encoding shRNA targeting WNT2B (TRCN0000033376 and TRCN0000033377), WNT9B (TRCN0000062069 and TRCN0000062070), WLS (TRCN0000133858 and TRCN0000133999), CTNNB1 (TRCN0000003843, TRCN0000003844 and TRCN0000003845) or shRNA non-target (NT, Sigma), 1.5 µg pMDLg/pRRE, 1.5 µg pRSV-REV and 3 µg pVSVg as previously described .

    Techniques: Genome Wide, Stable Transfection, Expressing, Luciferase, Transduction, shRNA, Transfection, Activity Assay, Quantitative RT-PCR, Infection, Functional Assay