post-transfection Search Results


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  • 86
    thermo fisher post transfection
    MMTV IRES activity is stimulated by S6K2-associated hnRNP A1 phosphorylations but inhibited by PRMT5 activity and PRMT5-induced hnRNP A1 methylations. ( A , B ) hnRNP A1-HA S4/S6 or S199 mutant plasmids (500 ng) were co-transfected with the dl MMTV IRES plasmid (300 ng). Total protein extracts were prepared 24 h <t>post-transfection.</t> ( A ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. ( B ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities obtained from the dl MMTV IRES vector in the presence of the wt-hnRNP A1-HA protein, set to 100%. Statistical analysis was performed by an ordinary one-way ANOVA test (* P
    Post Transfection, supplied by thermo fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore post transfection
    Expression and activity analysis of endogenously-tagged LRP6-mCherry in CRISPR/Cas9 edited NCI-H1703 cells and exogenous LRP6-mCherry in NCI-H1703 cells with stable gene insertion. ( a ) Confocal laser scanning microscopy images showing cell surface localization and relative expression levels of LRP6-mCherry in live cells. Non-specific autofluorescence is seen only adjacent to the nucleus (top panel with wt cells). ( b ) SDS-PAGE/western blot analysis showing relative expression levels of LRP6 receptor proteins in lysates from CRISPR/Cas9 and stable cell lines, compared to endogenous LRP6 from wt NCI-H1703 cells. Prior to harvest of cell lysates, cells were treated overnight with either control (con) CM or mWnt3a CM as indicated. Note the expected size increase of the LRP6 fusion protein upon tagging as well as the characteristic upshift of the LRP6 receptor protein upon Wnt stimulation, confirming Wnt-dependent receptor modification. The latter effect is less obvious in the stably expressing LRP6-mCherry cell line. Transferrin receptor (TfR) was used as a protein loading control. Transiently transfected cells were not included on the western blot as NCI-H1703 cell <t>transfection</t> efficiency is too low to allow comparison of average expression levels and instead requires analysis of single cells by microscopy. ( c ) TOPFLASH Wnt/β-catenin reporter assay showing the relative responsiveness of the LRP6-mCherry cell lines to both Wnt stimulation and DKK1-eGFP inhibition. Cells in 96-well plates were transfected with 20/5 ng of TOPFLASH/Renilla reporters, 8 ng mouse Wnt3a and 25 ng human DKK1-eGFP as indicated. 48 hr post transfection, cell lysates were harvested for luciferase assays. Note that both the CRISPR/Cas9 edited cell line and the cells stably expressing LRP6-mCherry respond to Wnt3a and DKK1 in the expected manner.
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    86
    Roche post transfection
    Genetic ablation of LGP 2 (encoded by the gene Dhx58 ) reveals ds RNA i in mammalian cells Dhx58 +/+ , Dhx58 +/− and Dhx58 −/− MEFs were transduced to stably express the d2GFP reporter. Cells were subsequently transfected with Cy5‐labelled dsRNA‐RL or dsRNA‐GFP and harvested 48 h <t>post‐transfection</t> to measure d2GFP expression in live, single, Cy5 + cells by flow cytometry. Histogram plots of one representative experiment are shown and are representative of four independent experiments with duplicate samples. Each histogram represents a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells. The median fluorescence values were normalised to those in Renilla‐transfected samples. Mean values and SD of four independent experiments with duplicate samples are shown. Statistical analysis was performed using one‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (** P
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    86
    Becton Dickinson post transfection
    Efficient p14-mediated fusion requires active adhesion. Fluorescent images (panels A–C) of QM5 fibroblasts fixed and stained with anti-N-cadherin antibody (green) or fluorescently conjugated phalloidin (red). Scale bars = 10 µm. (A) Cells cultured under low calcium conditions to disrupt cadherin-mediated cell–cell contacts and generate the no adhesion phenotype. (B) Cells cultured under low calcium conditions and then switched to normal calcium conditions to generate the active adhesion phenotype. (C) Cells cultured under low calcium conditions followed by treatment with cytoD under normal calcium conditions to allow cadherin engagement but not actin remodelling, generating the passive adhesion phenotype. Arrows in (B) indicate examples of F-actin and cadherin colocalization at sites of extended cell–cell contact. Arrows in panel (C) indicate sites of trans -cadherin interactions in the absence of extended junction formation. (D) QM5 cells were transfected with p14 and treated to generate the active, passive and no adhesion phenotypes depicted in the previous panels. Just prior to the onset of fusion (4 h <t>post-transfection),</t> cells were briefly subjected to calcium depletion (black and white bars) or maintained in normal calcium levels (grey bars). Immediately after depletion, cells were incubated in normal (white bars) or low calcium (black bars) medium in the presence or absence of cytoD as indicated. The extent of syncytium formation under the different culture conditions was quantified by syncytial indexing 2–4 h after the return to normal calcium containing media. Data is presented as the extent of fusion relative to cells maintained throughout the experiment in normal calcium medium with or without cytoD. Values represent the mean±S.E. (n = 5). The average numbers of syncytial nuclei per field under normal calcium levels (i.e. the 100% level) were 68.9 (no cytochalasin treatment) and 46.9 (with cytochalasin treatment).
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    Image Search Results


    MMTV IRES activity is stimulated by S6K2-associated hnRNP A1 phosphorylations but inhibited by PRMT5 activity and PRMT5-induced hnRNP A1 methylations. ( A , B ) hnRNP A1-HA S4/S6 or S199 mutant plasmids (500 ng) were co-transfected with the dl MMTV IRES plasmid (300 ng). Total protein extracts were prepared 24 h post-transfection. ( A ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. ( B ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities obtained from the dl MMTV IRES vector in the presence of the wt-hnRNP A1-HA protein, set to 100%. Statistical analysis was performed by an ordinary one-way ANOVA test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: MMTV IRES activity is stimulated by S6K2-associated hnRNP A1 phosphorylations but inhibited by PRMT5 activity and PRMT5-induced hnRNP A1 methylations. ( A , B ) hnRNP A1-HA S4/S6 or S199 mutant plasmids (500 ng) were co-transfected with the dl MMTV IRES plasmid (300 ng). Total protein extracts were prepared 24 h post-transfection. ( A ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. ( B ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities obtained from the dl MMTV IRES vector in the presence of the wt-hnRNP A1-HA protein, set to 100%. Statistical analysis was performed by an ordinary one-way ANOVA test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Activity Assay, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Expressing

    The hnRNP A1 knockdown negatively impacts on Gag protein synthesis from an HIV-1 molecular clone. ( A ) Schematic representation of the complete HIV-1 molecular clone pNL4.3-RLuc described in ( 51 ). ( B ) HEK 293T cells were co-transfected with the pNL4.3-RLuc (200 ng) plasmid and increasing concentrations (100, 150 or 250 nM) of a silencer RNA (siRNA) targeting the hnRNP A1 mRNA (siA1), or with a scrambled RNA (scRNA; 250 nM) as a control. The expression of the Gag-Rluc-HA fusion protein and hnRNP A1 was determined 48 h post-transfection by western blot, using the GAPDH protein as a loading control. The relative level of hnRNP A1 protein (%) was estimated based on the intensity of immunoreactive bands by imageJ, as detailed in Materials and Methods. ( C ) Cytoplasmic RNA was extracted from cells expressing pNL4.3-RLuc HIV-1 and treated with the scRNA (250 nM) or with the siA1 RNA (150 nM), and relative RNA levels were determined by real-time RT-qPCR. The RNA abundance was expressed relative to the value obtained for the cells treated with the scRNA set to 100%. Statistical analysis was performed by an unpaired two-tailed t-test . ( D ) Renilla luciferase activity was measured 48 h post-transfection and is expressed relative to the activity obtained when the pNL4.3-RLuc plasmid was co-transfected with the scRNA. Values represent the mean (±SEM) for three independent experiments, each conducted in duplicate. Statistical analysis was performed by an ordinary one-way ANOVA test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: The hnRNP A1 knockdown negatively impacts on Gag protein synthesis from an HIV-1 molecular clone. ( A ) Schematic representation of the complete HIV-1 molecular clone pNL4.3-RLuc described in ( 51 ). ( B ) HEK 293T cells were co-transfected with the pNL4.3-RLuc (200 ng) plasmid and increasing concentrations (100, 150 or 250 nM) of a silencer RNA (siRNA) targeting the hnRNP A1 mRNA (siA1), or with a scrambled RNA (scRNA; 250 nM) as a control. The expression of the Gag-Rluc-HA fusion protein and hnRNP A1 was determined 48 h post-transfection by western blot, using the GAPDH protein as a loading control. The relative level of hnRNP A1 protein (%) was estimated based on the intensity of immunoreactive bands by imageJ, as detailed in Materials and Methods. ( C ) Cytoplasmic RNA was extracted from cells expressing pNL4.3-RLuc HIV-1 and treated with the scRNA (250 nM) or with the siA1 RNA (150 nM), and relative RNA levels were determined by real-time RT-qPCR. The RNA abundance was expressed relative to the value obtained for the cells treated with the scRNA set to 100%. Statistical analysis was performed by an unpaired two-tailed t-test . ( D ) Renilla luciferase activity was measured 48 h post-transfection and is expressed relative to the activity obtained when the pNL4.3-RLuc plasmid was co-transfected with the scRNA. Values represent the mean (±SEM) for three independent experiments, each conducted in duplicate. Statistical analysis was performed by an ordinary one-way ANOVA test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, Luciferase, Activity Assay

    hnRNP A1 post-translational modifications impact on HIV-1 IRES activity. ( A ) hnRNP A1-HA mutants for the phosphorylation sites S4/S6 and S199, or methylation sites R218/R225 were generated by site-directed mutagenesis of the wt-hnRNP A1-HA template plasmid. ( B – E ) HEK 293T cells were co-transfected with the dl HIV-1 IRES (300 ng) together with each hnRNP A1-HA (500 ng) plasmid. Total protein extracts were prepared 24 h post-transfection. ( B , D ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. ( C , E ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities in the presence of the wt-hnRNP A1-HA, set to 100%. Values shown are the mean (±SEM) for three independent experiments, each performed in duplicate. Statistical analysis was performed by an unpaired two-tailed t -test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: hnRNP A1 post-translational modifications impact on HIV-1 IRES activity. ( A ) hnRNP A1-HA mutants for the phosphorylation sites S4/S6 and S199, or methylation sites R218/R225 were generated by site-directed mutagenesis of the wt-hnRNP A1-HA template plasmid. ( B – E ) HEK 293T cells were co-transfected with the dl HIV-1 IRES (300 ng) together with each hnRNP A1-HA (500 ng) plasmid. Total protein extracts were prepared 24 h post-transfection. ( B , D ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. ( C , E ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities in the presence of the wt-hnRNP A1-HA, set to 100%. Values shown are the mean (±SEM) for three independent experiments, each performed in duplicate. Statistical analysis was performed by an unpaired two-tailed t -test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Activity Assay, Methylation, Generated, Mutagenesis, Plasmid Preparation, Transfection, Western Blot, Expressing, Two Tailed Test

    Pharmacological inhibition of PRMT5 methyltransferase activity impairs HIV-1 IRES activity. ( A – C ) HEK 293T c ells were transfected with the dl HIV-1 IRES (300 ng) plasmid and treated, or not (only vehicle; (–)), with EPZ015666 (5, 10 or 20 μM), a specific inhibitor of the PRMT5 activity ( 57 ). Total protein extracts were prepared 48 h post-transfection and treatment. ( A ) The PRMT5 activity (SDMA), and levels of PRMT5 and hnRNP A1 proteins in extracts from untreated cell (lane 1) or from cells teated with at the different drug concentrations (5, 10, or 20 μM) were evaluated by western blot using GAPDH as a loading control. ( B , C ) RLuc and FLuc activities were measured, and data are presented as RLA ( B ) or RTA ( C ) relative to the non-treated (–) cells, set to 100%. Statistical analysis was performed by an ordinary two-way ( B ) or one-way ( C ) ANOVA test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: Pharmacological inhibition of PRMT5 methyltransferase activity impairs HIV-1 IRES activity. ( A – C ) HEK 293T c ells were transfected with the dl HIV-1 IRES (300 ng) plasmid and treated, or not (only vehicle; (–)), with EPZ015666 (5, 10 or 20 μM), a specific inhibitor of the PRMT5 activity ( 57 ). Total protein extracts were prepared 48 h post-transfection and treatment. ( A ) The PRMT5 activity (SDMA), and levels of PRMT5 and hnRNP A1 proteins in extracts from untreated cell (lane 1) or from cells teated with at the different drug concentrations (5, 10, or 20 μM) were evaluated by western blot using GAPDH as a loading control. ( B , C ) RLuc and FLuc activities were measured, and data are presented as RLA ( B ) or RTA ( C ) relative to the non-treated (–) cells, set to 100%. Statistical analysis was performed by an ordinary two-way ( B ) or one-way ( C ) ANOVA test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Inhibition, Activity Assay, Transfection, Plasmid Preparation, Western Blot

    hnRNP A1 does not enhance alternative splicing of the dl HIV-1 IRES RNA nor increases the cryptic promoter activity of the dl HIV-1 IRES DNA. ( A ) Schematic representation of the dl reporter targeted by the siRLuc a siRNA targeting the Renilla luciferase ORF. ( B , C ) The dl HIV-1 IRES (150 ng) was co-transfected with a control scRNA (100 nM) or with siRLuc (100 nM), in the presence, or the absence, of the wt-hnRNP A1-HA (250 ng) plasmid. Total protein extracts were prepared 48 hrs post-transfection. ( B ) The expression of the wt-hnRNP A1-HA recombinant protein was determined by western blot, using the GAPDH protein as a loading control. ( C ) RLuc and FLuc activities were measured and expressed as RLA relative to the values obtained with scRNA, set to 100%. ( D–F ) HEK 293T cells were transfected with either the dl HIV-1 IRES (150 ng) or a promoterless ΔSV40-dl HIV-1 IRES (150 ng) vector ( D ) in the presence, or the absence (-), of the wt-hnRNP A1-HA (250 ng) plasmid. 24 hrs post-transfection total protein extracts were prepared. ( E ) The expression of the hnRNP A1-HA recombinant protein was determined by western blot, using the GAPDH protein as a loading control. ( F ) RLuc and FLuc activities were measured, and results are expressed as RLA relative to the activities obtained from the dl HIV-1 IRES vector when in the absence of the wt-hnRNP A1-HA, set to 100%. Values shown are the mean (±SEM) for three independent experiments, each performed in duplicate. Statistical analysis was performed by an ordinary two-way ANOVA test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: hnRNP A1 does not enhance alternative splicing of the dl HIV-1 IRES RNA nor increases the cryptic promoter activity of the dl HIV-1 IRES DNA. ( A ) Schematic representation of the dl reporter targeted by the siRLuc a siRNA targeting the Renilla luciferase ORF. ( B , C ) The dl HIV-1 IRES (150 ng) was co-transfected with a control scRNA (100 nM) or with siRLuc (100 nM), in the presence, or the absence, of the wt-hnRNP A1-HA (250 ng) plasmid. Total protein extracts were prepared 48 hrs post-transfection. ( B ) The expression of the wt-hnRNP A1-HA recombinant protein was determined by western blot, using the GAPDH protein as a loading control. ( C ) RLuc and FLuc activities were measured and expressed as RLA relative to the values obtained with scRNA, set to 100%. ( D–F ) HEK 293T cells were transfected with either the dl HIV-1 IRES (150 ng) or a promoterless ΔSV40-dl HIV-1 IRES (150 ng) vector ( D ) in the presence, or the absence (-), of the wt-hnRNP A1-HA (250 ng) plasmid. 24 hrs post-transfection total protein extracts were prepared. ( E ) The expression of the hnRNP A1-HA recombinant protein was determined by western blot, using the GAPDH protein as a loading control. ( F ) RLuc and FLuc activities were measured, and results are expressed as RLA relative to the activities obtained from the dl HIV-1 IRES vector when in the absence of the wt-hnRNP A1-HA, set to 100%. Values shown are the mean (±SEM) for three independent experiments, each performed in duplicate. Statistical analysis was performed by an ordinary two-way ANOVA test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Recombinant, Western Blot

    HnRNP A1 is and ITAF for the HIV-1 IRES. ( A ) Schematic representation of dl HIV-1 IRES plasmid described in ( 7 ). The RLuc/FLuc bicistronic reporter mRNA is expressed from the SV40 promoter and receives a poly(A) tail by the SV40 poly(A) signal. The reporter mRNA harbors the 5′UTR of the HIV-1 mRNA (pNL4.3 HIV clone, GenBank accession number AF 324493 ). The reporter also contains the defective encephalomyocarditis virus (ΔEMCV) 5′UTR that lacks IRES activity but avoid ribosomal readthrough from the first cistron ( 7 , 85 ). ( B ) HEK 293T cells were co-transfected with the dl HIV-1 IRES (300 ng) and different quantities (25–500 ng) of a plasmid encoding for a wt-hnRNP A1-HA protein. The presence of the endogenous hnRNP A1 and overexpressed wt-hnRNP A1-HA proteins was confirmed by western blot using GAPDH as loading control. (C, D). RLuc and FLuc activities were measured 24 h post-transfection, and data are presented as relative luciferase activity (RLA) ( C ), or as relative translational activity (RTA) ( D ). RTA corresponds to the FLuc/RLuc ratio that is used as an index of IRES activity. The RLA and RTA values obtained in the absence of wt-hnRNP A1-HA plasmid were set to 100%. Statistical analysis was performed by an ordinary two-way ( C ) or one-way ( D ) ANOVA test. ( E , F ) The dl HIV-1 IRES plasmid (200 ng) was co-transfected with the siA1-RNA (150 nM) or with the scRNA (150 nM). ( E ) Western blots were performed to detect the expression level of hnRNP A1 (48 h post-transfection) using GAPDH as a loading control. Dilutions (%) of the protein extract generated from the scRNA transfected cells were used as a relative measure to quantify the impact of the siA1 RNA. ( F ) RLuc and Fluc activities were measured 48 h post-transfection, and data are presented as RLA (%), with the values obtained in the presence of the scRNA set to 100%. Values represent the mean (±SEM) for three independent experiments, each conducted in duplicate. Statistical analysis was performed by an unpaired two-tailed t -test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: HnRNP A1 is and ITAF for the HIV-1 IRES. ( A ) Schematic representation of dl HIV-1 IRES plasmid described in ( 7 ). The RLuc/FLuc bicistronic reporter mRNA is expressed from the SV40 promoter and receives a poly(A) tail by the SV40 poly(A) signal. The reporter mRNA harbors the 5′UTR of the HIV-1 mRNA (pNL4.3 HIV clone, GenBank accession number AF 324493 ). The reporter also contains the defective encephalomyocarditis virus (ΔEMCV) 5′UTR that lacks IRES activity but avoid ribosomal readthrough from the first cistron ( 7 , 85 ). ( B ) HEK 293T cells were co-transfected with the dl HIV-1 IRES (300 ng) and different quantities (25–500 ng) of a plasmid encoding for a wt-hnRNP A1-HA protein. The presence of the endogenous hnRNP A1 and overexpressed wt-hnRNP A1-HA proteins was confirmed by western blot using GAPDH as loading control. (C, D). RLuc and FLuc activities were measured 24 h post-transfection, and data are presented as relative luciferase activity (RLA) ( C ), or as relative translational activity (RTA) ( D ). RTA corresponds to the FLuc/RLuc ratio that is used as an index of IRES activity. The RLA and RTA values obtained in the absence of wt-hnRNP A1-HA plasmid were set to 100%. Statistical analysis was performed by an ordinary two-way ( C ) or one-way ( D ) ANOVA test. ( E , F ) The dl HIV-1 IRES plasmid (200 ng) was co-transfected with the siA1-RNA (150 nM) or with the scRNA (150 nM). ( E ) Western blots were performed to detect the expression level of hnRNP A1 (48 h post-transfection) using GAPDH as a loading control. Dilutions (%) of the protein extract generated from the scRNA transfected cells were used as a relative measure to quantify the impact of the siA1 RNA. ( F ) RLuc and Fluc activities were measured 48 h post-transfection, and data are presented as RLA (%), with the values obtained in the presence of the scRNA set to 100%. Values represent the mean (±SEM) for three independent experiments, each conducted in duplicate. Statistical analysis was performed by an unpaired two-tailed t -test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Plasmid Preparation, Activity Assay, Transfection, Western Blot, Luciferase, Expressing, Generated, Two Tailed Test

    HTLV-1 IRES activity is stimulated by PRMT5 activity and PRMT5-associated hnRNP A1 methylations. ( A-B ) hnRNP A1-HA mutant plasmids (500 ng) for the phosphorylation sites S4/S6 and S199 (indicated in the x-axis) were co-transfected with the dl HTLV-1 IRES plasmid (300 ng). Total protein extracts were prepared 24 hrs post-transfection. ( A ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. ( B ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities obtained from the dl HTLV-1 IRES vector in the presence of the wt-hnRNP A1 protein, set to 100%. Statistical analysis was performed by an ordinary one-way ANOVA test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: HTLV-1 IRES activity is stimulated by PRMT5 activity and PRMT5-associated hnRNP A1 methylations. ( A-B ) hnRNP A1-HA mutant plasmids (500 ng) for the phosphorylation sites S4/S6 and S199 (indicated in the x-axis) were co-transfected with the dl HTLV-1 IRES plasmid (300 ng). Total protein extracts were prepared 24 hrs post-transfection. ( A ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. ( B ) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities obtained from the dl HTLV-1 IRES vector in the presence of the wt-hnRNP A1 protein, set to 100%. Statistical analysis was performed by an ordinary one-way ANOVA test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Activity Assay, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Expressing

    HnRNP A1 is an ITAF for the MMTV IRES. ( A ) Schematic representation of the dl MMTV IRES plasmid described in ( 13 ). (B−D) HEK 293T cells were co-transfected with the dl MMTV IRES plasmid (300 ng) and increasing amounts (100, 250, 500 ng) of the wt-hnRNP A1-HA plasmid. Total protein extracts were prepared 24 hrs post-transfection. ( B ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. The relative level of hnRNP A1 protein (%) was estimated based on the intensity of immunoreactive bands by imageJ. ( C , D ) RLuc and FLuc activities were measured, and data are presented as RLA ( C ) or RTA ( D ). The RLA and RTA values in the absence of transfected wt-hnRNP A1-HA were set to 100%. Statistical analysis was performed by an ordinary two-way ( C ) or one-way ( D ) ANOVA test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: HnRNP A1 is an ITAF for the MMTV IRES. ( A ) Schematic representation of the dl MMTV IRES plasmid described in ( 13 ). (B−D) HEK 293T cells were co-transfected with the dl MMTV IRES plasmid (300 ng) and increasing amounts (100, 250, 500 ng) of the wt-hnRNP A1-HA plasmid. Total protein extracts were prepared 24 hrs post-transfection. ( B ) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. The relative level of hnRNP A1 protein (%) was estimated based on the intensity of immunoreactive bands by imageJ. ( C , D ) RLuc and FLuc activities were measured, and data are presented as RLA ( C ) or RTA ( D ). The RLA and RTA values in the absence of transfected wt-hnRNP A1-HA were set to 100%. Statistical analysis was performed by an ordinary two-way ( C ) or one-way ( D ) ANOVA test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Plasmid Preparation, Transfection, Western Blot, Expressing

    HnRNP A1 is an ITAF for the HTLV-1 IRES. ( A ) Schematic representation of the dl HTLV-1 IRES plasmid described in ( 12 ). ( B – D ) HEK 293T cells were co-transfected with the dl HTLV-1 IRES (300 ng) and increasing amounts of the wt-hnRNP A1-HA (100, 250, 500 ng) plasmid. Total protein extracts were prepared 24 h post-transfection. ( B ) Western blots were performed to follow the expression level of total hnRNP A1 and wt-hnRNP A-HA proteins using GAPDH as a loading control. The relative level of hnRNP A1 protein (%) was estimated based on the intensity of immunoreactive bands by imageJ. ( C , D ) RLuc and FLuc activities were measured, and data are presented as RLA ( C ) or RTA ( D ). The RLA and RTA values obtained when in the absence (–) of transfected wt-hnRNP A1-HA were set to 100%. Statistical analysis was performed by an ordinary two-way ( C ) or one-way ( D ) ANOVA test (* P

    Journal: Nucleic Acids Research

    Article Title: Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

    doi: 10.1093/nar/gkaa765

    Figure Lengend Snippet: HnRNP A1 is an ITAF for the HTLV-1 IRES. ( A ) Schematic representation of the dl HTLV-1 IRES plasmid described in ( 12 ). ( B – D ) HEK 293T cells were co-transfected with the dl HTLV-1 IRES (300 ng) and increasing amounts of the wt-hnRNP A1-HA (100, 250, 500 ng) plasmid. Total protein extracts were prepared 24 h post-transfection. ( B ) Western blots were performed to follow the expression level of total hnRNP A1 and wt-hnRNP A-HA proteins using GAPDH as a loading control. The relative level of hnRNP A1 protein (%) was estimated based on the intensity of immunoreactive bands by imageJ. ( C , D ) RLuc and FLuc activities were measured, and data are presented as RLA ( C ) or RTA ( D ). The RLA and RTA values obtained when in the absence (–) of transfected wt-hnRNP A1-HA were set to 100%. Statistical analysis was performed by an ordinary two-way ( C ) or one-way ( D ) ANOVA test (* P

    Article Snippet: Twenty-four hours post-transfection, cells were washed with PBS and resuspended in lysis buffer (NaCl 100 mM, EDTA 2 mM, Tris–HCl 50 mM pH 7.5, NaF 50 mM, sodium orthovanadate 1 mM, Triton X-100 1%, protease inhibitors (Roche Diagnostics).

    Techniques: Plasmid Preparation, Transfection, Western Blot, Expressing

    Expression and activity analysis of endogenously-tagged LRP6-mCherry in CRISPR/Cas9 edited NCI-H1703 cells and exogenous LRP6-mCherry in NCI-H1703 cells with stable gene insertion. ( a ) Confocal laser scanning microscopy images showing cell surface localization and relative expression levels of LRP6-mCherry in live cells. Non-specific autofluorescence is seen only adjacent to the nucleus (top panel with wt cells). ( b ) SDS-PAGE/western blot analysis showing relative expression levels of LRP6 receptor proteins in lysates from CRISPR/Cas9 and stable cell lines, compared to endogenous LRP6 from wt NCI-H1703 cells. Prior to harvest of cell lysates, cells were treated overnight with either control (con) CM or mWnt3a CM as indicated. Note the expected size increase of the LRP6 fusion protein upon tagging as well as the characteristic upshift of the LRP6 receptor protein upon Wnt stimulation, confirming Wnt-dependent receptor modification. The latter effect is less obvious in the stably expressing LRP6-mCherry cell line. Transferrin receptor (TfR) was used as a protein loading control. Transiently transfected cells were not included on the western blot as NCI-H1703 cell transfection efficiency is too low to allow comparison of average expression levels and instead requires analysis of single cells by microscopy. ( c ) TOPFLASH Wnt/β-catenin reporter assay showing the relative responsiveness of the LRP6-mCherry cell lines to both Wnt stimulation and DKK1-eGFP inhibition. Cells in 96-well plates were transfected with 20/5 ng of TOPFLASH/Renilla reporters, 8 ng mouse Wnt3a and 25 ng human DKK1-eGFP as indicated. 48 hr post transfection, cell lysates were harvested for luciferase assays. Note that both the CRISPR/Cas9 edited cell line and the cells stably expressing LRP6-mCherry respond to Wnt3a and DKK1 in the expected manner.

    Journal: eLife

    Article Title: Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

    doi: 10.7554/eLife.55286

    Figure Lengend Snippet: Expression and activity analysis of endogenously-tagged LRP6-mCherry in CRISPR/Cas9 edited NCI-H1703 cells and exogenous LRP6-mCherry in NCI-H1703 cells with stable gene insertion. ( a ) Confocal laser scanning microscopy images showing cell surface localization and relative expression levels of LRP6-mCherry in live cells. Non-specific autofluorescence is seen only adjacent to the nucleus (top panel with wt cells). ( b ) SDS-PAGE/western blot analysis showing relative expression levels of LRP6 receptor proteins in lysates from CRISPR/Cas9 and stable cell lines, compared to endogenous LRP6 from wt NCI-H1703 cells. Prior to harvest of cell lysates, cells were treated overnight with either control (con) CM or mWnt3a CM as indicated. Note the expected size increase of the LRP6 fusion protein upon tagging as well as the characteristic upshift of the LRP6 receptor protein upon Wnt stimulation, confirming Wnt-dependent receptor modification. The latter effect is less obvious in the stably expressing LRP6-mCherry cell line. Transferrin receptor (TfR) was used as a protein loading control. Transiently transfected cells were not included on the western blot as NCI-H1703 cell transfection efficiency is too low to allow comparison of average expression levels and instead requires analysis of single cells by microscopy. ( c ) TOPFLASH Wnt/β-catenin reporter assay showing the relative responsiveness of the LRP6-mCherry cell lines to both Wnt stimulation and DKK1-eGFP inhibition. Cells in 96-well plates were transfected with 20/5 ng of TOPFLASH/Renilla reporters, 8 ng mouse Wnt3a and 25 ng human DKK1-eGFP as indicated. 48 hr post transfection, cell lysates were harvested for luciferase assays. Note that both the CRISPR/Cas9 edited cell line and the cells stably expressing LRP6-mCherry respond to Wnt3a and DKK1 in the expected manner.

    Article Snippet: 24 hr post-transfection, the medium was exchanged, and 48 hr post-transfection one tenth of the cells were transferred to 10 cm2 dishes and cultured in RPMI 1640 supplemented with 700 µg/ml G418 (Sigma-Aldrich).

    Techniques: Expressing, Activity Assay, CRISPR, Confocal Laser Scanning Microscopy, SDS Page, Western Blot, Stable Transfection, Modification, Transfection, Microscopy, Reporter Assay, Inhibition, Luciferase

    Activity tests and western blot analysis of xKremen2-mCherry and xKremen2-eGFP. ( a ) TOPFLASH Wnt/β-catenin reporter assay. HEK293T cells in 96-wells were transfected with 25/5 ng TOPFLASH/Renilla reporters, 8 ng mWnt1 , 1 ng mFrizzled8 , 1 ng hDKK1-eGFP , 1 ng V5-xKremen2 , 1 ng V5-xKremen2-mCherry and 1 ng V5-xKremen2-eGFP as indicated. 24 hr post transfection, cell lysates were harvested for luciferase reporter assay. Note that, in the absence of DKK1-eGFP, the fluorescently tagged Kremen2 receptors show marginally greater inhibition of Wnt signaling compared to non-fluorescently tagged Kremen2 (compare lanes 4, 5 and 6). In the presence of DKK1, however, all Kremen2 constructs show a strong reduction (compare lane 3 with lanes 7, 8 and 9), confirming that they retain their ability to synergize with DKK1 in Wnt inhibition. ( b ) Western blot analysis to visualize the integrity of both V5-xKremen2-mCherry and V5-xKremen2-eGFP.

    Journal: eLife

    Article Title: Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

    doi: 10.7554/eLife.55286

    Figure Lengend Snippet: Activity tests and western blot analysis of xKremen2-mCherry and xKremen2-eGFP. ( a ) TOPFLASH Wnt/β-catenin reporter assay. HEK293T cells in 96-wells were transfected with 25/5 ng TOPFLASH/Renilla reporters, 8 ng mWnt1 , 1 ng mFrizzled8 , 1 ng hDKK1-eGFP , 1 ng V5-xKremen2 , 1 ng V5-xKremen2-mCherry and 1 ng V5-xKremen2-eGFP as indicated. 24 hr post transfection, cell lysates were harvested for luciferase reporter assay. Note that, in the absence of DKK1-eGFP, the fluorescently tagged Kremen2 receptors show marginally greater inhibition of Wnt signaling compared to non-fluorescently tagged Kremen2 (compare lanes 4, 5 and 6). In the presence of DKK1, however, all Kremen2 constructs show a strong reduction (compare lane 3 with lanes 7, 8 and 9), confirming that they retain their ability to synergize with DKK1 in Wnt inhibition. ( b ) Western blot analysis to visualize the integrity of both V5-xKremen2-mCherry and V5-xKremen2-eGFP.

    Article Snippet: 24 hr post-transfection, the medium was exchanged, and 48 hr post-transfection one tenth of the cells were transferred to 10 cm2 dishes and cultured in RPMI 1640 supplemented with 700 µg/ml G418 (Sigma-Aldrich).

    Techniques: Activity Assay, Western Blot, Reporter Assay, Transfection, Luciferase, Inhibition, Construct

    Activity tests and western blot analysis of DKK1-eGFP and DKK2-eGFP. ( a ) NCI-H1703 cells in 96-well format were transfected with 20/2 ng TOPFLASH/Renilla reporters. 16 hr post transfection, mWnt3a CM was added for 6 hr, followed by a 12 hr treatment with either DKK1-eGFP or DKK2-eGFP CM, as indicated, before cell lysates were harvested for the luciferase assay. ( b ) Western blot analysis to visualize the integrity of the DKK1-eGFP and DKK2-eGFP fusion proteins present in the CM. The DKK1 and DKK2 fusion proteins show major bands at ~68 and~58 kDa, respectively. The expected molecular weights are 56 kDa for DKK1-eGFP and 55 kDa for DKK2-eGFP, suggesting that DKK1-eGFP has more post-translational modification(s), which is consistent with the appearance of additional bands for DKK1-eGFP. The amounts of original CM loaded were 0.33 µl for DKK1-eGFP and 10 µl for DKK2-eGFP.

    Journal: eLife

    Article Title: Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

    doi: 10.7554/eLife.55286

    Figure Lengend Snippet: Activity tests and western blot analysis of DKK1-eGFP and DKK2-eGFP. ( a ) NCI-H1703 cells in 96-well format were transfected with 20/2 ng TOPFLASH/Renilla reporters. 16 hr post transfection, mWnt3a CM was added for 6 hr, followed by a 12 hr treatment with either DKK1-eGFP or DKK2-eGFP CM, as indicated, before cell lysates were harvested for the luciferase assay. ( b ) Western blot analysis to visualize the integrity of the DKK1-eGFP and DKK2-eGFP fusion proteins present in the CM. The DKK1 and DKK2 fusion proteins show major bands at ~68 and~58 kDa, respectively. The expected molecular weights are 56 kDa for DKK1-eGFP and 55 kDa for DKK2-eGFP, suggesting that DKK1-eGFP has more post-translational modification(s), which is consistent with the appearance of additional bands for DKK1-eGFP. The amounts of original CM loaded were 0.33 µl for DKK1-eGFP and 10 µl for DKK2-eGFP.

    Article Snippet: 24 hr post-transfection, the medium was exchanged, and 48 hr post-transfection one tenth of the cells were transferred to 10 cm2 dishes and cultured in RPMI 1640 supplemented with 700 µg/ml G418 (Sigma-Aldrich).

    Techniques: Activity Assay, Western Blot, Transfection, Luciferase, Modification

    siRNA-mediated knockdown to characterize CRISPR/Cas9 edited NCI-H1703 cells. ( a, b ) TOPFLASH Wnt reporter assays showing the effect of siRNA-mediated silencing of all LRP6 receptors (siLRP6) or only the endogenously tagged LRP6 receptors (siCherry/siTomato). CRISPR/Cas9 edited NCI-H1703 cells expressing endogenously-tagged LRP6-mCherry ( a ) and LRP6-tdTomato ( b ) as well as wt cells ( a and b ) were co-transfected in 96-well format with 3.3 pmol siRNA (targeting either LRP6, mCherry or tdTomato) and 25/5 ng TOPFLASH/Renilla reporters. 18 hr post-transfection, cells were treated overnight with control or Wnt3a CM before harvesting cell lysates for luciferase assays. For each treatment, luciferase values were set to one for the control samples in order to compare Wnt responsiveness between cell lines. As expected, knockdown of all LRP6 receptors with siLRP6 resulted in a strong reduction of the Wnt-induced TOPFLASH signal, although the LRP6-tdTomato CRISPR cell line displayed a somewhat weaker reduction. For specific knockdown of the endogenously tagged LRP6-mCherry species using siCherry, the degree of Wnt activation was significantly reduced when compared to the effect of siCherry on the corresponding wt cells. This partial reduction confirms that LRP6-mCherry contributes to Wnt signaling activity seen in these cells but that siRNA resistant, non-tagged LRP6 remains. Similarly, for LRP6-tdTomato silencing using siTomato, a significantly reduced Wnt activation was observed when compared to the effect of siTomato on wt cells. These results confirm that LRP6-mCherry and LRP6-tdTomato are active in Wnt reception, and, in agreement with our sequencing results of the CRISPR/Cas9 cell lines, that both cell lines are heterozygous, such that only one of the LRP6 alleles has been endogenously tagged. ( c ) Western blot analysis of LRP6, LRP6-mCherry and LRP6-tdTomato from wt and CRISPR/Cas9 edited NCI-H1703 cell lines after the indicated siRNA transfections. Cells were transfected with 39.6 pmol siRNA in 24-well format and cell lysates were harvested after 48 hr for Western blot analysis. Note that knockdown using siLRP6 leads to complete loss of all LRP6 receptor proteins (lanes 2, 6 and 9), whereas knockdown using siCherry (lane 7) or LRP6-tdTomato (lane 10) results in specific loss of the endogenously tagged LRP6 bands with higher molecular weight, and not the lower molecular weight wt LRP6 bands. These results further confirm that the CRISPR/Cas9 edited cell lines are heterozygous with respect to targeted endogenous tagging of LRP6. The band intensities also confirm that the endogenously tagged LRP6 receptors are expressed at higher levels than their corresponding wt receptors. Vinculin was used as a loading control.

    Journal: eLife

    Article Title: Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

    doi: 10.7554/eLife.55286

    Figure Lengend Snippet: siRNA-mediated knockdown to characterize CRISPR/Cas9 edited NCI-H1703 cells. ( a, b ) TOPFLASH Wnt reporter assays showing the effect of siRNA-mediated silencing of all LRP6 receptors (siLRP6) or only the endogenously tagged LRP6 receptors (siCherry/siTomato). CRISPR/Cas9 edited NCI-H1703 cells expressing endogenously-tagged LRP6-mCherry ( a ) and LRP6-tdTomato ( b ) as well as wt cells ( a and b ) were co-transfected in 96-well format with 3.3 pmol siRNA (targeting either LRP6, mCherry or tdTomato) and 25/5 ng TOPFLASH/Renilla reporters. 18 hr post-transfection, cells were treated overnight with control or Wnt3a CM before harvesting cell lysates for luciferase assays. For each treatment, luciferase values were set to one for the control samples in order to compare Wnt responsiveness between cell lines. As expected, knockdown of all LRP6 receptors with siLRP6 resulted in a strong reduction of the Wnt-induced TOPFLASH signal, although the LRP6-tdTomato CRISPR cell line displayed a somewhat weaker reduction. For specific knockdown of the endogenously tagged LRP6-mCherry species using siCherry, the degree of Wnt activation was significantly reduced when compared to the effect of siCherry on the corresponding wt cells. This partial reduction confirms that LRP6-mCherry contributes to Wnt signaling activity seen in these cells but that siRNA resistant, non-tagged LRP6 remains. Similarly, for LRP6-tdTomato silencing using siTomato, a significantly reduced Wnt activation was observed when compared to the effect of siTomato on wt cells. These results confirm that LRP6-mCherry and LRP6-tdTomato are active in Wnt reception, and, in agreement with our sequencing results of the CRISPR/Cas9 cell lines, that both cell lines are heterozygous, such that only one of the LRP6 alleles has been endogenously tagged. ( c ) Western blot analysis of LRP6, LRP6-mCherry and LRP6-tdTomato from wt and CRISPR/Cas9 edited NCI-H1703 cell lines after the indicated siRNA transfections. Cells were transfected with 39.6 pmol siRNA in 24-well format and cell lysates were harvested after 48 hr for Western blot analysis. Note that knockdown using siLRP6 leads to complete loss of all LRP6 receptor proteins (lanes 2, 6 and 9), whereas knockdown using siCherry (lane 7) or LRP6-tdTomato (lane 10) results in specific loss of the endogenously tagged LRP6 bands with higher molecular weight, and not the lower molecular weight wt LRP6 bands. These results further confirm that the CRISPR/Cas9 edited cell lines are heterozygous with respect to targeted endogenous tagging of LRP6. The band intensities also confirm that the endogenously tagged LRP6 receptors are expressed at higher levels than their corresponding wt receptors. Vinculin was used as a loading control.

    Article Snippet: 24 hr post-transfection, the medium was exchanged, and 48 hr post-transfection one tenth of the cells were transferred to 10 cm2 dishes and cultured in RPMI 1640 supplemented with 700 µg/ml G418 (Sigma-Aldrich).

    Techniques: CRISPR, Expressing, Transfection, Luciferase, Activation Assay, Activity Assay, Sequencing, Western Blot, Molecular Weight

    Genetic ablation of LGP 2 (encoded by the gene Dhx58 ) reveals ds RNA i in mammalian cells Dhx58 +/+ , Dhx58 +/− and Dhx58 −/− MEFs were transduced to stably express the d2GFP reporter. Cells were subsequently transfected with Cy5‐labelled dsRNA‐RL or dsRNA‐GFP and harvested 48 h post‐transfection to measure d2GFP expression in live, single, Cy5 + cells by flow cytometry. Histogram plots of one representative experiment are shown and are representative of four independent experiments with duplicate samples. Each histogram represents a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells. The median fluorescence values were normalised to those in Renilla‐transfected samples. Mean values and SD of four independent experiments with duplicate samples are shown. Statistical analysis was performed using one‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (** P

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: Genetic ablation of LGP 2 (encoded by the gene Dhx58 ) reveals ds RNA i in mammalian cells Dhx58 +/+ , Dhx58 +/− and Dhx58 −/− MEFs were transduced to stably express the d2GFP reporter. Cells were subsequently transfected with Cy5‐labelled dsRNA‐RL or dsRNA‐GFP and harvested 48 h post‐transfection to measure d2GFP expression in live, single, Cy5 + cells by flow cytometry. Histogram plots of one representative experiment are shown and are representative of four independent experiments with duplicate samples. Each histogram represents a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells. The median fluorescence values were normalised to those in Renilla‐transfected samples. Mean values and SD of four independent experiments with duplicate samples are shown. Statistical analysis was performed using one‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (** P

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: Stable Transfection, Transfection, Expressing, Flow Cytometry, Cytometry, Fluorescence

    Set up of the in vitro dicing assays and related controls Purification of FLAG‐tagged human Dicer from HEK293T cells. FLAG‐hDicer was expressed in HEK293T cells by transient transfection and subsequently immunoprecipitated using a FLAG antibody, followed by three rounds of elution from the resin using 3FLAG‐peptide. Aliquots were analysed by SDS–PAGE and Coomassie staining. The remaining fraction on the beads was tested to verify efficient elution. The small RNAs generated in the dicing assay require the catalytic activity of Dicer. Fifty nM dsRNA internally labelled with Cy5 was incubated with 500 nM wild‐type FLAG‐hDicer or a catalytic mutant (D1320A/D1709A) for 1 h at 37°C prior to analysis on a denaturing polyacrylamide gel by in‐gel fluorescence. To verify equal protein input into the dicing assay of Fig 4 C, an equal amount of protein was taken for analysis by SDS–PAGE and immunoblotting using a FLAG antibody. To verify equal protein input into the dicing assay of Fig 4 D, a small aliquot of each reaction was analysed by SDS–PAGE and immunoblotting using a FLAG antibody.

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: Set up of the in vitro dicing assays and related controls Purification of FLAG‐tagged human Dicer from HEK293T cells. FLAG‐hDicer was expressed in HEK293T cells by transient transfection and subsequently immunoprecipitated using a FLAG antibody, followed by three rounds of elution from the resin using 3FLAG‐peptide. Aliquots were analysed by SDS–PAGE and Coomassie staining. The remaining fraction on the beads was tested to verify efficient elution. The small RNAs generated in the dicing assay require the catalytic activity of Dicer. Fifty nM dsRNA internally labelled with Cy5 was incubated with 500 nM wild‐type FLAG‐hDicer or a catalytic mutant (D1320A/D1709A) for 1 h at 37°C prior to analysis on a denaturing polyacrylamide gel by in‐gel fluorescence. To verify equal protein input into the dicing assay of Fig 4 C, an equal amount of protein was taken for analysis by SDS–PAGE and immunoblotting using a FLAG antibody. To verify equal protein input into the dicing assay of Fig 4 D, a small aliquot of each reaction was analysed by SDS–PAGE and immunoblotting using a FLAG antibody.

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: In Vitro, Purification, Transfection, Immunoprecipitation, SDS Page, Staining, Generated, Activity Assay, Incubation, Mutagenesis, Fluorescence

    Analysis of doxycycline‐inducible expression of full‐length FLAG‐LGP2, FLAG‐LGP2 CTD, or FLAG‐LGP2 CTD K634E in Ifnar1 −/− MEFs Relative expression (RE) of LGP2 ( DHX58 ) in wild‐type MEFs treated for 24 or 48 h with type I IFN was assessed by qRT–PCR and normalised to β‐actin ( ACTB ) using the ∆∆ C t method. Verification of doxycycline‐dependent induction of FLAG‐LGP2 expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iLGP2 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Verification of doxycycline‐dependent induction of FLAG‐LGP2 CTD and CTD K634E expression in Ifnar1 −/− MEFs. The indicated clones were treated for 72 h with dox and subsequently fixed, permeabilised, stained with a FLAG‐Cy3 antibody and analysed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐tagged human LGP2 CTD or CTD K634E is induced following 72 h of dox treatment. β‐Actin serves as loading control. Northern blot analysis of dsRNA‐derived siRNAs in two individual clones of Ifnar1 −/− iLGP2 CTD and CTD K634E MEFs left untreated or treated with dox for 24 h prior to transfection with dsRNA‐GFP. Twenty‐four hours post‐transfection, cells were harvested and the generation of siRNAs was analysed by Northern blotting using a probe specific for dsRNA‐GFP. The arrow points to dsRNA‐GFP‐derived siRNAs. A miRNA ladder was used as a size marker and endogenous U6 served as loading control. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: Analysis of doxycycline‐inducible expression of full‐length FLAG‐LGP2, FLAG‐LGP2 CTD, or FLAG‐LGP2 CTD K634E in Ifnar1 −/− MEFs Relative expression (RE) of LGP2 ( DHX58 ) in wild‐type MEFs treated for 24 or 48 h with type I IFN was assessed by qRT–PCR and normalised to β‐actin ( ACTB ) using the ∆∆ C t method. Verification of doxycycline‐dependent induction of FLAG‐LGP2 expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iLGP2 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Verification of doxycycline‐dependent induction of FLAG‐LGP2 CTD and CTD K634E expression in Ifnar1 −/− MEFs. The indicated clones were treated for 72 h with dox and subsequently fixed, permeabilised, stained with a FLAG‐Cy3 antibody and analysed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐tagged human LGP2 CTD or CTD K634E is induced following 72 h of dox treatment. β‐Actin serves as loading control. Northern blot analysis of dsRNA‐derived siRNAs in two individual clones of Ifnar1 −/− iLGP2 CTD and CTD K634E MEFs left untreated or treated with dox for 24 h prior to transfection with dsRNA‐GFP. Twenty‐four hours post‐transfection, cells were harvested and the generation of siRNAs was analysed by Northern blotting using a probe specific for dsRNA‐GFP. The arrow points to dsRNA‐GFP‐derived siRNAs. A miRNA ladder was used as a size marker and endogenous U6 served as loading control. Source data are available online for this figure.

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Clone Assay, Staining, Northern Blot, Derivative Assay, Transfection, Marker

    Expression of RIG‐I or MDA5, unlike that of LGP2, does not inhibit dsRNA‐mediated RNAi in Ifnar1 −/− cells Doxycycline treatment does not impact on dsRNAi in parental Ifnar1 −/− MEFs that lack inducible LGP2 expression constructs. Verification of doxycycline‐dependent induction of FLAG‐RIG‐I (iRIG‐I) and FLAG‐MDA5 (iMDA5) expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iRIG‐I and iMDA5 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐RIG‐I or FLAG‐MDA5 is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. Expression of full‐length RIG‐I or MDA5 does not affect dsRNA‐mediated RNAi in Ifnar1 −/− cells. Ifnar1 −/− iRIG‐I and Ifnar1 −/− iMDA5 cells, which also express a destabilised form of GFP (d2GFP), were transfected with Cy5‐labelled long dsRNA corresponding to the first 200 nt of Renilla luciferase (dsRNA‐RL) or GFP (dsRNA‐GFP) in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of one representative clone are shown and are representative of three independent experiments. Each histogram and bar represents a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. The median fluorescence values were normalised to those in Renilla‐transfected samples. Mean values and SD of three independent experiments are shown. Statistical analysis was performed using two‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (ns, not significant). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: Expression of RIG‐I or MDA5, unlike that of LGP2, does not inhibit dsRNA‐mediated RNAi in Ifnar1 −/− cells Doxycycline treatment does not impact on dsRNAi in parental Ifnar1 −/− MEFs that lack inducible LGP2 expression constructs. Verification of doxycycline‐dependent induction of FLAG‐RIG‐I (iRIG‐I) and FLAG‐MDA5 (iMDA5) expression in Ifnar1 −/− MEFs by flow cytometry. Various Ifnar1 −/− iRIG‐I and iMDA5 clones were treated for 72 h with doxycycline (dox) and subsequently fixed, permeabilised and stained with a FLAG‐Cy3 antibody followed by flow cytometry. Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐RIG‐I or FLAG‐MDA5 is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. Expression of full‐length RIG‐I or MDA5 does not affect dsRNA‐mediated RNAi in Ifnar1 −/− cells. Ifnar1 −/− iRIG‐I and Ifnar1 −/− iMDA5 cells, which also express a destabilised form of GFP (d2GFP), were transfected with Cy5‐labelled long dsRNA corresponding to the first 200 nt of Renilla luciferase (dsRNA‐RL) or GFP (dsRNA‐GFP) in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of one representative clone are shown and are representative of three independent experiments. Each histogram and bar represents a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. The median fluorescence values were normalised to those in Renilla‐transfected samples. Mean values and SD of three independent experiments are shown. Statistical analysis was performed using two‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (ns, not significant). Source data are available online for this figure.

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: Expressing, Construct, Flow Cytometry, Cytometry, Clone Assay, Staining, Transfection, Luciferase, Fluorescence

    Dicer and its co‐factors do not impact on the ability of LGP 2 to bind viral RNA and induce MDA 5‐dependent type I IFN signalling HEK293 cells stably expressing FLAG‐LGP2 were treated with siRNAs targeting MDA5, TRBP, PACT or Dicer or non‐targeting control siRNAs. Knockdown efficiency was determined by SDS–PAGE and immunoblotting using the indicated antibodies (left panel). β‐Actin serves as loading control. Three days post‐transfection, the IFN response to the indicated doses of total RNA extracted from EMCV‐infected HeLa cells was tested in an IFN‐β promotor luciferase‐based reporter assay (right panel). Mean values and SEM of three independent experiments are shown. Statistical analysis was performed using one‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons using untransfected cells as control for each dose. Significant differences were observed for siMDA5 only (* P

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: Dicer and its co‐factors do not impact on the ability of LGP 2 to bind viral RNA and induce MDA 5‐dependent type I IFN signalling HEK293 cells stably expressing FLAG‐LGP2 were treated with siRNAs targeting MDA5, TRBP, PACT or Dicer or non‐targeting control siRNAs. Knockdown efficiency was determined by SDS–PAGE and immunoblotting using the indicated antibodies (left panel). β‐Actin serves as loading control. Three days post‐transfection, the IFN response to the indicated doses of total RNA extracted from EMCV‐infected HeLa cells was tested in an IFN‐β promotor luciferase‐based reporter assay (right panel). Mean values and SEM of three independent experiments are shown. Statistical analysis was performed using one‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons using untransfected cells as control for each dose. Significant differences were observed for siMDA5 only (* P

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: Multiple Displacement Amplification, Stable Transfection, Expressing, SDS Page, Transfection, Infection, Luciferase, Reporter Assay

    Expression of full‐length LGP 2 inhibits ds RNA ‐mediated RNA i in Ifnar1 −/− cells Ifnar1 −/− iLGP2 cells, which also express a destabilised form of GFP (d2GFP), were transfected with Cy5‐labelled long dsRNA corresponding to the first 200 nt of Renilla luciferase (dsRNA‐RL) or GFP (dsRNA‐GFP) in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of two representative clones are shown. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. Unlike full‐length LGP2, LGP2 CTD or CTD K634E expression is unable to suppress dsRNAi in Ifnar1 −/− MEFs. Four independent clones of Ifnar1 −/− d2GFP MEFs that inducibly express LGP2 CTD or LGP2 K634E were treated with dsRNA‐RL or dsRNA‐GFP as in (A). Histogram plot of one representative clone for each construct is shown. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. Data information: All histogram plots are representative of three independent experiments. Each histogram represents a sample size of 10,000 cells. Bar graphs depict the median fluorescence values normalised to those in Renilla‐transfected samples. Mean values and SD of three independent experiments are shown. Statistical analysis was performed using two‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (ns, not significant; * P

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: Expression of full‐length LGP 2 inhibits ds RNA ‐mediated RNA i in Ifnar1 −/− cells Ifnar1 −/− iLGP2 cells, which also express a destabilised form of GFP (d2GFP), were transfected with Cy5‐labelled long dsRNA corresponding to the first 200 nt of Renilla luciferase (dsRNA‐RL) or GFP (dsRNA‐GFP) in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of two representative clones are shown. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. Unlike full‐length LGP2, LGP2 CTD or CTD K634E expression is unable to suppress dsRNAi in Ifnar1 −/− MEFs. Four independent clones of Ifnar1 −/− d2GFP MEFs that inducibly express LGP2 CTD or LGP2 K634E were treated with dsRNA‐RL or dsRNA‐GFP as in (A). Histogram plot of one representative clone for each construct is shown. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells in four independent clones. Data information: All histogram plots are representative of three independent experiments. Each histogram represents a sample size of 10,000 cells. Bar graphs depict the median fluorescence values normalised to those in Renilla‐transfected samples. Mean values and SD of three independent experiments are shown. Statistical analysis was performed using two‐way ANOVA with Sidak's multiple comparisons test as post‐test for pairwise comparisons. Significant differences with Sidak's multiple comparisons test are shown (ns, not significant; * P

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: Expressing, Transfection, Luciferase, Flow Cytometry, Cytometry, Clone Assay, Fluorescence, Construct

    Expression of LGP 2 does not affect ds RNA ‐mediated RNA i in F9 embryonic carcinoma cells Immunoblot analysis of F9 embryonic carcinoma cells in which expression of FLAG‐tagged human LGP2 is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. F9 iLGP2 cells were stably transduced with a lentivirus encoding a destabilised form of GFP (d2GFP) and subsequently transfected with Cy5‐labelled dsRNA‐RL or dsRNA‐GFP in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of one representative experiment are shown and represent a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells. Mean values and SD of two independent experiments with duplicate samples are shown. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: Expression of LGP 2 does not affect ds RNA ‐mediated RNA i in F9 embryonic carcinoma cells Immunoblot analysis of F9 embryonic carcinoma cells in which expression of FLAG‐tagged human LGP2 is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. F9 iLGP2 cells were stably transduced with a lentivirus encoding a destabilised form of GFP (d2GFP) and subsequently transfected with Cy5‐labelled dsRNA‐RL or dsRNA‐GFP in the absence or presence or doxycycline. Forty‐eight hours post‐transfection, cells were harvested and d2GFP expression in live, single, Cy5 + cells was analysed by flow cytometry. Histogram plots of one representative experiment are shown and represent a sample size of 10,000 cells. Bar graphs display the percentage of GFP median fluorescence intensity of dsRNA‐GFP‐transfected cells relative to dsRNA‐RL‐transfected cells. Mean values and SD of two independent experiments with duplicate samples are shown. Source data are available online for this figure.

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: Expressing, Stable Transfection, Transduction, Transfection, Flow Cytometry, Cytometry, Fluorescence

    LGP 2 is sufficient to inhibit ds RNA processing by Dicer in cells lacking a competent type I IFN system Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐tagged human LGP2 (iLGP2) is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. Northern blot analysis of dsRNA‐derived siRNAs in Ifnar1 −/− iLGP2 MEFs left untreated or treated with dox for 24 h prior to transfection with dsRNA‐GFP. Twenty‐four hours post‐transfection, cells were harvested and the generation of siRNAs was analysed by Northern blotting using a probe specific for dsRNA‐GFP. The arrow points to dsRNA‐GFP‐derived siRNAs. RNA from an in vitro dicing reaction using dsRNA‐GFP was loaded in parallel. A miRNA ladder was used as a size marker, and endogenous U6 served as loading control. The membrane was first probed for dsRNA‐GFP, then stripped and reprobed for the miRNA marker, U6 and miR‐16. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

    doi: 10.15252/embj.201797479

    Figure Lengend Snippet: LGP 2 is sufficient to inhibit ds RNA processing by Dicer in cells lacking a competent type I IFN system Immunoblot analysis of four clones of Ifnar1 −/− MEFs in which expression of FLAG‐tagged human LGP2 (iLGP2) is induced following 72 h of doxycycline (dox) treatment. β‐Actin serves as loading control. Northern blot analysis of dsRNA‐derived siRNAs in Ifnar1 −/− iLGP2 MEFs left untreated or treated with dox for 24 h prior to transfection with dsRNA‐GFP. Twenty‐four hours post‐transfection, cells were harvested and the generation of siRNAs was analysed by Northern blotting using a probe specific for dsRNA‐GFP. The arrow points to dsRNA‐GFP‐derived siRNAs. RNA from an in vitro dicing reaction using dsRNA‐GFP was loaded in parallel. A miRNA ladder was used as a size marker, and endogenous U6 served as loading control. The membrane was first probed for dsRNA‐GFP, then stripped and reprobed for the miRNA marker, U6 and miR‐16. Source data are available online for this figure.

    Article Snippet: Twenty‐eight hours post‐transfection, cells were lysed in lysis buffer [30 mM Tris pH 6.8, 50 mM NaCl, 3 mM MgCl2 , 5% glycerol, 0.4% NP‐40 and protease inhibitors (Roche)], and the lysate was cleared by centrifugation.

    Techniques: Clone Assay, Expressing, Northern Blot, Derivative Assay, Transfection, In Vitro, Marker

    Efficient p14-mediated fusion requires active adhesion. Fluorescent images (panels A–C) of QM5 fibroblasts fixed and stained with anti-N-cadherin antibody (green) or fluorescently conjugated phalloidin (red). Scale bars = 10 µm. (A) Cells cultured under low calcium conditions to disrupt cadherin-mediated cell–cell contacts and generate the no adhesion phenotype. (B) Cells cultured under low calcium conditions and then switched to normal calcium conditions to generate the active adhesion phenotype. (C) Cells cultured under low calcium conditions followed by treatment with cytoD under normal calcium conditions to allow cadherin engagement but not actin remodelling, generating the passive adhesion phenotype. Arrows in (B) indicate examples of F-actin and cadherin colocalization at sites of extended cell–cell contact. Arrows in panel (C) indicate sites of trans -cadherin interactions in the absence of extended junction formation. (D) QM5 cells were transfected with p14 and treated to generate the active, passive and no adhesion phenotypes depicted in the previous panels. Just prior to the onset of fusion (4 h post-transfection), cells were briefly subjected to calcium depletion (black and white bars) or maintained in normal calcium levels (grey bars). Immediately after depletion, cells were incubated in normal (white bars) or low calcium (black bars) medium in the presence or absence of cytoD as indicated. The extent of syncytium formation under the different culture conditions was quantified by syncytial indexing 2–4 h after the return to normal calcium containing media. Data is presented as the extent of fusion relative to cells maintained throughout the experiment in normal calcium medium with or without cytoD. Values represent the mean±S.E. (n = 5). The average numbers of syncytial nuclei per field under normal calcium levels (i.e. the 100% level) were 68.9 (no cytochalasin treatment) and 46.9 (with cytochalasin treatment).

    Journal: PLoS Pathogens

    Article Title: A Virus-Encoded Cell-Cell Fusion Machine Dependent on Surrogate Adhesins

    doi: 10.1371/journal.ppat.1000016

    Figure Lengend Snippet: Efficient p14-mediated fusion requires active adhesion. Fluorescent images (panels A–C) of QM5 fibroblasts fixed and stained with anti-N-cadherin antibody (green) or fluorescently conjugated phalloidin (red). Scale bars = 10 µm. (A) Cells cultured under low calcium conditions to disrupt cadherin-mediated cell–cell contacts and generate the no adhesion phenotype. (B) Cells cultured under low calcium conditions and then switched to normal calcium conditions to generate the active adhesion phenotype. (C) Cells cultured under low calcium conditions followed by treatment with cytoD under normal calcium conditions to allow cadherin engagement but not actin remodelling, generating the passive adhesion phenotype. Arrows in (B) indicate examples of F-actin and cadherin colocalization at sites of extended cell–cell contact. Arrows in panel (C) indicate sites of trans -cadherin interactions in the absence of extended junction formation. (D) QM5 cells were transfected with p14 and treated to generate the active, passive and no adhesion phenotypes depicted in the previous panels. Just prior to the onset of fusion (4 h post-transfection), cells were briefly subjected to calcium depletion (black and white bars) or maintained in normal calcium levels (grey bars). Immediately after depletion, cells were incubated in normal (white bars) or low calcium (black bars) medium in the presence or absence of cytoD as indicated. The extent of syncytium formation under the different culture conditions was quantified by syncytial indexing 2–4 h after the return to normal calcium containing media. Data is presented as the extent of fusion relative to cells maintained throughout the experiment in normal calcium medium with or without cytoD. Values represent the mean±S.E. (n = 5). The average numbers of syncytial nuclei per field under normal calcium levels (i.e. the 100% level) were 68.9 (no cytochalasin treatment) and 46.9 (with cytochalasin treatment).

    Article Snippet: Cells were subjected to calcium depletion at 3 h post-transfection, just prior to the onset of syncytiogenesis, and incubated in either MEM or S-MEM for an additional 3 h. Cells were trypsinized, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur (Becton Dickinson)) using appropriate filter sets and Cell Quest software.

    Techniques: Staining, Cell Culture, Transfection, Incubation

    Calcium depletion inhibits FAST protein-mediated syncytiogenesis. (A) QM5 and MDCK cells were transfected with p14, cultured under normal or low calcium conditions, then fixed and Giemsa stained at 8 (QM5) or 20 (MDCK) h post-transfection to detect multinucleated syncytia. Scale bar = 50 µm. (B) Influenza HA- (HA), VSV G- (G) and FAST protein (p10, p14, p15) -transfected QM5 fibroblasts, and p14-transfected MDCK (MD) or HT1080 (HT) epithelial cells were cultured under normal or low calcium conditions. Cells were fixed at various times post-transfection when syncytia were countable, and the average number of syncytial nuclei per field was determined from Giemsa-stained monolayers. Data is presented as the extent of cell fusion under low calcium conditions relative to the same transfected cells cultured for the same length of time under normal calcium conditions, set at 100%. Values represent the mean±S.E. (n = 3). The following numbers are the average syncytial nuclei per field for the different fusogens in the different cell lines under normal calcium levels (i.e. the 100% level): p14 in MDCK cells, 20 h post-transfection - 81.2; p14 in HT1080 cells, 9 h post-transfection - 44.9; p14 in QM5 cells, 7 h post-transfection - 200.4; p10 in QM5 cells, 9 h post-transfection - 83.1; p15 in QM5 cells, 13 h post-transfection - 101.6; VSV-G protein in QM5 cells, 24 h post-transfection, no low pH treatment - 60.9; influenza HA in QM5 cells, low pH treatment at 24 h post-transfection - 293.11.

    Journal: PLoS Pathogens

    Article Title: A Virus-Encoded Cell-Cell Fusion Machine Dependent on Surrogate Adhesins

    doi: 10.1371/journal.ppat.1000016

    Figure Lengend Snippet: Calcium depletion inhibits FAST protein-mediated syncytiogenesis. (A) QM5 and MDCK cells were transfected with p14, cultured under normal or low calcium conditions, then fixed and Giemsa stained at 8 (QM5) or 20 (MDCK) h post-transfection to detect multinucleated syncytia. Scale bar = 50 µm. (B) Influenza HA- (HA), VSV G- (G) and FAST protein (p10, p14, p15) -transfected QM5 fibroblasts, and p14-transfected MDCK (MD) or HT1080 (HT) epithelial cells were cultured under normal or low calcium conditions. Cells were fixed at various times post-transfection when syncytia were countable, and the average number of syncytial nuclei per field was determined from Giemsa-stained monolayers. Data is presented as the extent of cell fusion under low calcium conditions relative to the same transfected cells cultured for the same length of time under normal calcium conditions, set at 100%. Values represent the mean±S.E. (n = 3). The following numbers are the average syncytial nuclei per field for the different fusogens in the different cell lines under normal calcium levels (i.e. the 100% level): p14 in MDCK cells, 20 h post-transfection - 81.2; p14 in HT1080 cells, 9 h post-transfection - 44.9; p14 in QM5 cells, 7 h post-transfection - 200.4; p10 in QM5 cells, 9 h post-transfection - 83.1; p15 in QM5 cells, 13 h post-transfection - 101.6; VSV-G protein in QM5 cells, 24 h post-transfection, no low pH treatment - 60.9; influenza HA in QM5 cells, low pH treatment at 24 h post-transfection - 293.11.

    Article Snippet: Cells were subjected to calcium depletion at 3 h post-transfection, just prior to the onset of syncytiogenesis, and incubated in either MEM or S-MEM for an additional 3 h. Cells were trypsinized, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur (Becton Dickinson)) using appropriate filter sets and Cell Quest software.

    Techniques: Transfection, Cell Culture, Staining

    Effects of cytoD on QM5 cell adhesion and p14-induced syncytiogenesis. (A) Fluorescent images of QM5 fibroblasts treated with 0 (a), 0.5 (b) or 1 µg/ml (c) of cytoD for 30 minutes prior to fixation and staining with fluorescently conjugated phalloidin (green) and propidium iodide (red). Scale bars = 10 µm. (B) Cell–cell fusion in QM5 cells treated with the indicated concentrations of cytoD was quantified at 7 h post-transfection by syncytial indexing. Data is presented as the extent of fusion relative to cells untreated with cytoD, set at 100%. Values represent the mean±S.E. (n = 3). (C) QM5 fibroblasts transfected with p14 and left untreated (top panel) or treated with 0.1 µg/ml of cytoD for 1 h (bottom panel) were surface immunostained using an anti-p14 ectodomain-specific antiserum and Alexa Fluor 488-conjugated secondary antibody (red line tracing) and analyzed for cell-surface fluorescence (in arbitrary units) by flow cytometry relative to mock-transfected cells (grey histogram).

    Journal: PLoS Pathogens

    Article Title: A Virus-Encoded Cell-Cell Fusion Machine Dependent on Surrogate Adhesins

    doi: 10.1371/journal.ppat.1000016

    Figure Lengend Snippet: Effects of cytoD on QM5 cell adhesion and p14-induced syncytiogenesis. (A) Fluorescent images of QM5 fibroblasts treated with 0 (a), 0.5 (b) or 1 µg/ml (c) of cytoD for 30 minutes prior to fixation and staining with fluorescently conjugated phalloidin (green) and propidium iodide (red). Scale bars = 10 µm. (B) Cell–cell fusion in QM5 cells treated with the indicated concentrations of cytoD was quantified at 7 h post-transfection by syncytial indexing. Data is presented as the extent of fusion relative to cells untreated with cytoD, set at 100%. Values represent the mean±S.E. (n = 3). (C) QM5 fibroblasts transfected with p14 and left untreated (top panel) or treated with 0.1 µg/ml of cytoD for 1 h (bottom panel) were surface immunostained using an anti-p14 ectodomain-specific antiserum and Alexa Fluor 488-conjugated secondary antibody (red line tracing) and analyzed for cell-surface fluorescence (in arbitrary units) by flow cytometry relative to mock-transfected cells (grey histogram).

    Article Snippet: Cells were subjected to calcium depletion at 3 h post-transfection, just prior to the onset of syncytiogenesis, and incubated in either MEM or S-MEM for an additional 3 h. Cells were trypsinized, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur (Becton Dickinson)) using appropriate filter sets and Cell Quest software.

    Techniques: Staining, Transfection, Fluorescence, Flow Cytometry, Cytometry

    Surrogate receptor binding proteins enhance p14-mediated fusion in the absence of cadherin junctions. (A) QM5 and QM5-HAO cells were transfected with p14 and cell–cell fusion under normal (black bars) or low (white bars) calcium conditions in the presence of FBS was quantified by syncytial indexing at 7 h post-transfection. QM5-HAO cells were also transfected under similar conditions and incubated in the presence of horse serum (HS) to block HAO receptor binding, or were transfected with the non-fusogenic mutant p14-G2A or empty vector (no p14) to assess any contribution of the HAO protein to syncytiogenesis. Data is presented as the extent of fusion under low calcium conditions relative to normal calcium conditions. Values represent the mean±S.E. (n = 3–5). The average numbers of syncytial nuclei per field under normal calcium levels (i.e. the 100% level) were 151.9 (QM5 cells in FBS), 169.4 (QM5-HA0 cells in FBS), and 51.2 (QM5-HA0 cells in HS). (B) Merged surface immunofluorescence microscopy images of epitope-tagged p14 (red) and influenza HA (green) in QM5-HAO cells under normal and low calcium conditions. Yellow indicates regions of p14 and HAO colocalization. Scale bars = 10 µm.

    Journal: PLoS Pathogens

    Article Title: A Virus-Encoded Cell-Cell Fusion Machine Dependent on Surrogate Adhesins

    doi: 10.1371/journal.ppat.1000016

    Figure Lengend Snippet: Surrogate receptor binding proteins enhance p14-mediated fusion in the absence of cadherin junctions. (A) QM5 and QM5-HAO cells were transfected with p14 and cell–cell fusion under normal (black bars) or low (white bars) calcium conditions in the presence of FBS was quantified by syncytial indexing at 7 h post-transfection. QM5-HAO cells were also transfected under similar conditions and incubated in the presence of horse serum (HS) to block HAO receptor binding, or were transfected with the non-fusogenic mutant p14-G2A or empty vector (no p14) to assess any contribution of the HAO protein to syncytiogenesis. Data is presented as the extent of fusion under low calcium conditions relative to normal calcium conditions. Values represent the mean±S.E. (n = 3–5). The average numbers of syncytial nuclei per field under normal calcium levels (i.e. the 100% level) were 151.9 (QM5 cells in FBS), 169.4 (QM5-HA0 cells in FBS), and 51.2 (QM5-HA0 cells in HS). (B) Merged surface immunofluorescence microscopy images of epitope-tagged p14 (red) and influenza HA (green) in QM5-HAO cells under normal and low calcium conditions. Yellow indicates regions of p14 and HAO colocalization. Scale bars = 10 µm.

    Article Snippet: Cells were subjected to calcium depletion at 3 h post-transfection, just prior to the onset of syncytiogenesis, and incubated in either MEM or S-MEM for an additional 3 h. Cells were trypsinized, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur (Becton Dickinson)) using appropriate filter sets and Cell Quest software.

    Techniques: Binding Assay, Transfection, Incubation, Blocking Assay, Mutagenesis, Plasmid Preparation, Immunofluorescence, Microscopy

    Ectopic cadherin expression enhances p14-mediated fusion. (A) Phase contrast microscopy (400× mag.) of L and EL cells. (B) L and EL cells were transfected with p14, cultured under normal (black bars) or low (white bars) calcium conditions, and the extent of syncytia formation in Giemsa-stained monolayers was quantified at 26 h post-transfection by syncytial indexing. Data is presented as the extent of fusion relative to L cells under normal calcium conditions. Values represent the mean±S.E. (n = 3). The average number of syncytial nuclei per field in the L cells under normal calcium levels (i.e. the 100% level) was 40.7.

    Journal: PLoS Pathogens

    Article Title: A Virus-Encoded Cell-Cell Fusion Machine Dependent on Surrogate Adhesins

    doi: 10.1371/journal.ppat.1000016

    Figure Lengend Snippet: Ectopic cadherin expression enhances p14-mediated fusion. (A) Phase contrast microscopy (400× mag.) of L and EL cells. (B) L and EL cells were transfected with p14, cultured under normal (black bars) or low (white bars) calcium conditions, and the extent of syncytia formation in Giemsa-stained monolayers was quantified at 26 h post-transfection by syncytial indexing. Data is presented as the extent of fusion relative to L cells under normal calcium conditions. Values represent the mean±S.E. (n = 3). The average number of syncytial nuclei per field in the L cells under normal calcium levels (i.e. the 100% level) was 40.7.

    Article Snippet: Cells were subjected to calcium depletion at 3 h post-transfection, just prior to the onset of syncytiogenesis, and incubated in either MEM or S-MEM for an additional 3 h. Cells were trypsinized, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur (Becton Dickinson)) using appropriate filter sets and Cell Quest software.

    Techniques: Expressing, Microscopy, Transfection, Cell Culture, Staining

    Ablating cadherin expression inhibits p14-mediated syncytiogenesis. (A) Transfected QM5 cells were surface immunostained for p14 (green), then permeabilized and immunostained for N-cadherin (red) at 4 h post-transfection. Arrows in the merged image point to regions of N-cadherin and p14 co-localization at cell–cell contacts as indicated by the yellow pixels. The differential interference microscopy (DIC) image of the same field is shown. Scale bar = 10 µm. (B) HT-1080 cells were transfected with control siRNA or siRNA directed against human N-cadherin, then co-transfected with p14 and cultured under normal (black bars) or low (white bars) calcium conditions. Cells were fixed at 9 h post p14-transfection and syncytiogenesis was quantified by syncytial indexing. Data is presented as the extent of fusion relative to control siRNA-transfected cells cultured under normal calcium conditions. Values represent the mean±S.E. (n = 4). The average number of syncytial nuclei per field under normal calcium levels (i.e. the 100% level) was 53.8. The inset shows Western blot analysis of N-cadherin (N-Cad) and actin expression in the cells transfected with control (Cont) or N-cadherin (N-Cad) siRNA. Numbers indicate the mobilities of M r standards.

    Journal: PLoS Pathogens

    Article Title: A Virus-Encoded Cell-Cell Fusion Machine Dependent on Surrogate Adhesins

    doi: 10.1371/journal.ppat.1000016

    Figure Lengend Snippet: Ablating cadherin expression inhibits p14-mediated syncytiogenesis. (A) Transfected QM5 cells were surface immunostained for p14 (green), then permeabilized and immunostained for N-cadherin (red) at 4 h post-transfection. Arrows in the merged image point to regions of N-cadherin and p14 co-localization at cell–cell contacts as indicated by the yellow pixels. The differential interference microscopy (DIC) image of the same field is shown. Scale bar = 10 µm. (B) HT-1080 cells were transfected with control siRNA or siRNA directed against human N-cadherin, then co-transfected with p14 and cultured under normal (black bars) or low (white bars) calcium conditions. Cells were fixed at 9 h post p14-transfection and syncytiogenesis was quantified by syncytial indexing. Data is presented as the extent of fusion relative to control siRNA-transfected cells cultured under normal calcium conditions. Values represent the mean±S.E. (n = 4). The average number of syncytial nuclei per field under normal calcium levels (i.e. the 100% level) was 53.8. The inset shows Western blot analysis of N-cadherin (N-Cad) and actin expression in the cells transfected with control (Cont) or N-cadherin (N-Cad) siRNA. Numbers indicate the mobilities of M r standards.

    Article Snippet: Cells were subjected to calcium depletion at 3 h post-transfection, just prior to the onset of syncytiogenesis, and incubated in either MEM or S-MEM for an additional 3 h. Cells were trypsinized, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur (Becton Dickinson)) using appropriate filter sets and Cell Quest software.

    Techniques: Expressing, Transfection, Microscopy, Cell Culture, Western Blot

    Fusion efficiencies in cadherin-containing and cadherin-deficient cell types. (A) QM5 and L cells were transfected with p14 or VSV G, cultured under normal calcium conditions, then fixed and Giemsa stained at 8 (panel a) or 24 (panels b–d) h post-transfection to detect multinucleated syncytia. Scale bar = 50 µm. (B) QM5 and L cells were transfected with VSV G or p14, cultured under normal calcium conditions, and the average number of syncytial nuclei per field was determined from Giemsa-stained monolayers at the indicated times post-transfection. Values represent the mean±S.D. from a representative of two separate experiments conducted in triplicate.

    Journal: PLoS Pathogens

    Article Title: A Virus-Encoded Cell-Cell Fusion Machine Dependent on Surrogate Adhesins

    doi: 10.1371/journal.ppat.1000016

    Figure Lengend Snippet: Fusion efficiencies in cadherin-containing and cadherin-deficient cell types. (A) QM5 and L cells were transfected with p14 or VSV G, cultured under normal calcium conditions, then fixed and Giemsa stained at 8 (panel a) or 24 (panels b–d) h post-transfection to detect multinucleated syncytia. Scale bar = 50 µm. (B) QM5 and L cells were transfected with VSV G or p14, cultured under normal calcium conditions, and the average number of syncytial nuclei per field was determined from Giemsa-stained monolayers at the indicated times post-transfection. Values represent the mean±S.D. from a representative of two separate experiments conducted in triplicate.

    Article Snippet: Cells were subjected to calcium depletion at 3 h post-transfection, just prior to the onset of syncytiogenesis, and incubated in either MEM or S-MEM for an additional 3 h. Cells were trypsinized, resuspended in PBS, and analyzed by flow cytometry (FACSCalibur (Becton Dickinson)) using appropriate filter sets and Cell Quest software.

    Techniques: Transfection, Cell Culture, Staining