xbp1 gene  (New England Biolabs)


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    EnGen Mutation Detection Kit
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    EnGen Mutation Detection Kit 25 rxns
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    New England Biolabs xbp1 gene
    EnGen Mutation Detection Kit
    EnGen Mutation Detection Kit 25 rxns
    https://www.bioz.com/result/xbp1 gene/product/New England Biolabs
    Average 80 stars, based on 18476 article reviews
    Price from $9.99 to $1999.99
    xbp1 gene - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections"

    Article Title: The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15844-2

    XBP1s binds to predicted sites on the AdV E1A and E4-e/p. a Schematics depicting the XBP1-binding sites (blue and red boxes) and consensus-binding sequences on the E1A, E4, and major late (ML) promoters in AdV-C5 genome. The letter psi denotes the packaging sequence near the inverted terminal repeat (ITR). The yellow box on the right shows the conservation of predicted XBP1s-binding sites in the E1A and E4 promoters in different human AdV types. b Chromatin immunoprecipitations from AdV-C2-infected cells using anti-XBP1s and control IgG antibodies. The data show the fold enrichment of XBP1s on the AdV-C2 promoters (in particular the E1A and the E4 promoters) with anti-XBP1s versus control antibodies, as calculated using precleared chromatin as input 36 . Data show the means from three technical replicates. Two independent ChIP experiments gave similar results. c The deletion and mutagenesis of the XBP1s-binding sites on the E1A enhancer/promoter (e/p), nucleotide positions 63–195 reduce the E1A expression in normal cells and cells ectopically expressing XBP1s. E1A expression analyses in control or hXBP1s-transduced HDF-TERT cells infected with C5-dl309 or the mutant virus C5-dl309-∆63–195, and E1A expression analysis in AdV-C5 and mutant (mut) AdV-C5-XBP1s-mut-infected HDF-TERT cells (MOI 75000). Eight thousand cells per condition, including non-infected cells (n.i.), were chosen by random sampling of the total data. The mutagenized E1A-e/p region in AdV-C5-XBP1s-mut virus is shown on the right side with A
    Figure Legend Snippet: XBP1s binds to predicted sites on the AdV E1A and E4-e/p. a Schematics depicting the XBP1-binding sites (blue and red boxes) and consensus-binding sequences on the E1A, E4, and major late (ML) promoters in AdV-C5 genome. The letter psi denotes the packaging sequence near the inverted terminal repeat (ITR). The yellow box on the right shows the conservation of predicted XBP1s-binding sites in the E1A and E4 promoters in different human AdV types. b Chromatin immunoprecipitations from AdV-C2-infected cells using anti-XBP1s and control IgG antibodies. The data show the fold enrichment of XBP1s on the AdV-C2 promoters (in particular the E1A and the E4 promoters) with anti-XBP1s versus control antibodies, as calculated using precleared chromatin as input 36 . Data show the means from three technical replicates. Two independent ChIP experiments gave similar results. c The deletion and mutagenesis of the XBP1s-binding sites on the E1A enhancer/promoter (e/p), nucleotide positions 63–195 reduce the E1A expression in normal cells and cells ectopically expressing XBP1s. E1A expression analyses in control or hXBP1s-transduced HDF-TERT cells infected with C5-dl309 or the mutant virus C5-dl309-∆63–195, and E1A expression analysis in AdV-C5 and mutant (mut) AdV-C5-XBP1s-mut-infected HDF-TERT cells (MOI 75000). Eight thousand cells per condition, including non-infected cells (n.i.), were chosen by random sampling of the total data. The mutagenized E1A-e/p region in AdV-C5-XBP1s-mut virus is shown on the right side with A

    Techniques Used: Binding Assay, Sequencing, Infection, Chromatin Immunoprecipitation, Mutagenesis, Expressing, Sampling

    IRE1α and XBP1s facilitate persistent and lytic AdV infections. a Reduction in late AdV-C5 protein VI expression (MOI 180) of HDF-TERT cells upon RNA interference against IRE1α and XBP1 in the presence or absence of IFN-γ, including nontargeting siRNA (siNeg1). Data show the means ± SD from three independent experiments ( n = 3). b E1A expression of AdV-C5-infected HDF-TERT cells (MOI 200, 37 °C, 1 h) with or without 500 IU IFN-γ, or IRE1α RNase inhibitor 4µ8C (100 µM) at 13 days pi was analyzed by immunofluorescence showing representative images (left) and a scatterplot with 15,000 cells per condition (middle). Data show the median, first and third quartiles, and whiskers as boxes and lines, respectively. Significance was assessed with two-tailed Wilcoxon nonparametric test (middle panels). Virus titers (q-PCR, right panel) were determined after 5 days of incubation with the drug (18 d pi). Q-PCR data show the means from two technical replicates. Two independent experiments gave similar results. Scale bar, 100 µm. c xCELLigence impedance plots showing HDF-TERT cell viability upon dl309 or dl309-63/195 and AdV-C5 or AdV-C5-XBP1s-mut infections (MOI 200). Cells were seeded on xCELLigence E-16 plate, and impedance readout for cell viability was measured live at 15-min intervals. Data show the means ± SD from three technical replicates. Two independent experiments gave similar results (left row). Experimental conditions and virus amount were as in panel b Genome copy numbers of virions released to the supernatant from the same experiment. Data show the means from two technical replicates. Two independent experiments gave similar results (middle row). Representative phase-contrast images of parallel samples imaged live for dl309 or dl309-63/195 infections (images on the right side, scale bar, 200 µm). d Schematic model depicting AdV infection under the control of a five-component feedforward loop. (1) The immediate early E1A protein transactivates early promoters. including the E3 and E4, giving rise to the 19K glycoprotein (2). Activation of IRE1α by 19 K increases XBP1s mRNA, and XBP1s protein (3), which translocates into the nucleus, and binds to the E1A enhancer/promoter (e/p) of the episomal viral genome (4). Binding of XBP1s to the E1A-e/p increases the E1A levels (5), which enhances output from the E3 promoter, enhances the 19K levels, and maintains a feedforward loop supporting viral persistence and lytic infection.
    Figure Legend Snippet: IRE1α and XBP1s facilitate persistent and lytic AdV infections. a Reduction in late AdV-C5 protein VI expression (MOI 180) of HDF-TERT cells upon RNA interference against IRE1α and XBP1 in the presence or absence of IFN-γ, including nontargeting siRNA (siNeg1). Data show the means ± SD from three independent experiments ( n = 3). b E1A expression of AdV-C5-infected HDF-TERT cells (MOI 200, 37 °C, 1 h) with or without 500 IU IFN-γ, or IRE1α RNase inhibitor 4µ8C (100 µM) at 13 days pi was analyzed by immunofluorescence showing representative images (left) and a scatterplot with 15,000 cells per condition (middle). Data show the median, first and third quartiles, and whiskers as boxes and lines, respectively. Significance was assessed with two-tailed Wilcoxon nonparametric test (middle panels). Virus titers (q-PCR, right panel) were determined after 5 days of incubation with the drug (18 d pi). Q-PCR data show the means from two technical replicates. Two independent experiments gave similar results. Scale bar, 100 µm. c xCELLigence impedance plots showing HDF-TERT cell viability upon dl309 or dl309-63/195 and AdV-C5 or AdV-C5-XBP1s-mut infections (MOI 200). Cells were seeded on xCELLigence E-16 plate, and impedance readout for cell viability was measured live at 15-min intervals. Data show the means ± SD from three technical replicates. Two independent experiments gave similar results (left row). Experimental conditions and virus amount were as in panel b Genome copy numbers of virions released to the supernatant from the same experiment. Data show the means from two technical replicates. Two independent experiments gave similar results (middle row). Representative phase-contrast images of parallel samples imaged live for dl309 or dl309-63/195 infections (images on the right side, scale bar, 200 µm). d Schematic model depicting AdV infection under the control of a five-component feedforward loop. (1) The immediate early E1A protein transactivates early promoters. including the E3 and E4, giving rise to the 19K glycoprotein (2). Activation of IRE1α by 19 K increases XBP1s mRNA, and XBP1s protein (3), which translocates into the nucleus, and binds to the E1A enhancer/promoter (e/p) of the episomal viral genome (4). Binding of XBP1s to the E1A-e/p increases the E1A levels (5), which enhances output from the E3 promoter, enhances the 19K levels, and maintains a feedforward loop supporting viral persistence and lytic infection.

    Techniques Used: Expressing, Infection, Immunofluorescence, Two Tailed Test, Polymerase Chain Reaction, Incubation, Activation Assay, Binding Assay

    IRE1α activation enhances AdV infection of HeLa cells. a IRE1α-knockout (I-KO) HeLa cells are less susceptible to infection by AdV-C5 (MOI 75, 75 vp/cell) compared with normal HeLa, as indicated by late viral protein VI expression, whereas the ectopic lentivirus-mediated expression of IRE1α in HeLa I-KO cells restores infection (left panel, upper row middle and right panels showing representative images and quantifications, respectively. Scale bar, 200 µm). Data show the means ± SD from four independent experiments. Reduced virus growth in HeLa I-KO cells compared with wild-type cells. Cells were infected with AdV-C5 (MOI 500) for 1 h at 37 °C (equivalent to MOI 50 in continuous infection), unbound virus washed off, and virus titers from cells and supernatant measured at 48 and 72 hpi (lower row middle panel). Data show the means from two independent experiments. The IRE1α endonuclease inhibitor 4µ8C reduces AdV-C5 titers in long-term infections of HDF-TERT cells (lower row, right panel). HDF-TERT cells were infected with AdV-C5 (MOI 200, 37 °C, 1 h), followed by addition of 4µ8C (100 µM) 13 days pi. Data show the means from two independent experiments. b . IRE1α and the expression of the viral E1A protein are required to enhance XBP1 splicing in AdV-C2 and C5 infections. Rescue of XBP1 splicing in AdV-infected I-KO cells by IRE1α overexpression (first panel). Cells were transduced and infected as in a ; cell lysates were subjected to XBP1 splicing assays at 24 hpi. Reduced XBP1s transcripts in HeLa I-KO compared with normal HeLa cells upon AdV-C5 infection (MOI 5, second panel). Data show the means ± SD from three independent experiments. XBP1 splicing in human conjunctival epithelial cells infected with AdV-C2 or C5 (MOI 75, third panel). E1A -deleted AdV-C5 mutant does not activate XBP1 splicing in HeLa cells 24 hpi (MOI 200 each, fourth panel). The asterisk denotes a background product. Source data are provided as a Source Data file.
    Figure Legend Snippet: IRE1α activation enhances AdV infection of HeLa cells. a IRE1α-knockout (I-KO) HeLa cells are less susceptible to infection by AdV-C5 (MOI 75, 75 vp/cell) compared with normal HeLa, as indicated by late viral protein VI expression, whereas the ectopic lentivirus-mediated expression of IRE1α in HeLa I-KO cells restores infection (left panel, upper row middle and right panels showing representative images and quantifications, respectively. Scale bar, 200 µm). Data show the means ± SD from four independent experiments. Reduced virus growth in HeLa I-KO cells compared with wild-type cells. Cells were infected with AdV-C5 (MOI 500) for 1 h at 37 °C (equivalent to MOI 50 in continuous infection), unbound virus washed off, and virus titers from cells and supernatant measured at 48 and 72 hpi (lower row middle panel). Data show the means from two independent experiments. The IRE1α endonuclease inhibitor 4µ8C reduces AdV-C5 titers in long-term infections of HDF-TERT cells (lower row, right panel). HDF-TERT cells were infected with AdV-C5 (MOI 200, 37 °C, 1 h), followed by addition of 4µ8C (100 µM) 13 days pi. Data show the means from two independent experiments. b . IRE1α and the expression of the viral E1A protein are required to enhance XBP1 splicing in AdV-C2 and C5 infections. Rescue of XBP1 splicing in AdV-infected I-KO cells by IRE1α overexpression (first panel). Cells were transduced and infected as in a ; cell lysates were subjected to XBP1 splicing assays at 24 hpi. Reduced XBP1s transcripts in HeLa I-KO compared with normal HeLa cells upon AdV-C5 infection (MOI 5, second panel). Data show the means ± SD from three independent experiments. XBP1 splicing in human conjunctival epithelial cells infected with AdV-C2 or C5 (MOI 75, third panel). E1A -deleted AdV-C5 mutant does not activate XBP1 splicing in HeLa cells 24 hpi (MOI 200 each, fourth panel). The asterisk denotes a background product. Source data are provided as a Source Data file.

    Techniques Used: Activation Assay, Infection, Knock-Out, Expressing, Over Expression, Mutagenesis

    AdV infection of mouse embryonic fibroblasts induces XBP1 splicing and displaces BiP/Grp78 from IRE1α. a Flag-IRE1α-expressing MEFs but not IRE1α-KO MEFs induce XBP1s upon AdV-C2 infection (MOI 300) or thapsigargin (Tg, 10 µM) treatment (left panel). Representative immunofluorescence images with the anti-19K antibody 3A9 showing 19K expression in AdV-C-infected IRE1α-KO MEFs and Flag-IRE1α-expressing IRE1α-KO MEFs 24 hpi (clones 30 and 574, respectively, right panel, scale bar, 20 µm). Three independent experiments gave similar results. Source data are provided as a Source Data file. b AdV induces phosphorylation of IRE1α. Flag-IRE1α expressing IRE1α-KO MEFs at 7, 16, and 24 hpi with AdV-C5 (MOI 300) immunoblotted with anti-IRE1α antibody. Lysates were resolved on a 6% SDS-PAGE gel containing 25 µM Phos-tag. Samples were treated with or without alkaline phosphatase. Phosphorylated (p) and hypophosphorylated (o) forms of IRE1α are indicated by the dashed lines, and the percentage of IRE1α phosphorylated was calculated as indicated. Lysates are the same as in panel D demonstrating β-tubulin loading. Three independent experiments gave similar results. Source data are provided as a Source Data file. c AdV-C5 infection of HeLa cells (MOI 200) does not activate PERK, unlike treatment of cells with the reducing agent DTT. Activated phosphorylated PERK is indicated by p, and the inactive form by o. Two independent experiments gave similar results. d BiP displacement from IRE1α occurs before XBP1 splicing in Flag-IRE1α-expressing IRE1α-KO MEFs infected with AdV-C5 (MOI 300). Cells were lysed and BiP–IRE1α complexes immunoprecipitated (IP) with anti-Flag antibody, and a western blot with anti-IRE1α, anti-BiP, and anti-β-tubulin antibodies was performed. A separate non-reducing immunoblot probed with anti-19K Tw1.3 antibodies revealed monomeric and dimer forms of 19K indicated as mo and di, respectively. Input lysates were 1% of the immunoprecipitated samples. The corresponding samples were also analyzed for XBP1 splicing and E1A mRNA levels by rt-PCR (reverse transcription polymerase chain reaction), as indicated. Three independent experiments gave similar results. Source data are provided as a Source Data file. e BiP–IRE1α dissociation requires E1, not 19K. Co-immunoprecipitation of Flag-IRE1α and BiP was performed as described in d with AdV mutants lacking E1 (AdV-C5-∆E1) and 19K (AdV-C5-Δ19K) at MOI 300 each. Two independent experiments gave similar results. Source data are provided as a Source Data file. f AdV-C5 infection does not increase BiP/Grp78 and IRE1α levels given in arbitrary units (a.u.), unlike the canonical UPR triggered by DTT (2 mM). The bar graph shows the normalized levels of IRE1α and BiP from three independent experiments. Data show the means ± SD from three independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: AdV infection of mouse embryonic fibroblasts induces XBP1 splicing and displaces BiP/Grp78 from IRE1α. a Flag-IRE1α-expressing MEFs but not IRE1α-KO MEFs induce XBP1s upon AdV-C2 infection (MOI 300) or thapsigargin (Tg, 10 µM) treatment (left panel). Representative immunofluorescence images with the anti-19K antibody 3A9 showing 19K expression in AdV-C-infected IRE1α-KO MEFs and Flag-IRE1α-expressing IRE1α-KO MEFs 24 hpi (clones 30 and 574, respectively, right panel, scale bar, 20 µm). Three independent experiments gave similar results. Source data are provided as a Source Data file. b AdV induces phosphorylation of IRE1α. Flag-IRE1α expressing IRE1α-KO MEFs at 7, 16, and 24 hpi with AdV-C5 (MOI 300) immunoblotted with anti-IRE1α antibody. Lysates were resolved on a 6% SDS-PAGE gel containing 25 µM Phos-tag. Samples were treated with or without alkaline phosphatase. Phosphorylated (p) and hypophosphorylated (o) forms of IRE1α are indicated by the dashed lines, and the percentage of IRE1α phosphorylated was calculated as indicated. Lysates are the same as in panel D demonstrating β-tubulin loading. Three independent experiments gave similar results. Source data are provided as a Source Data file. c AdV-C5 infection of HeLa cells (MOI 200) does not activate PERK, unlike treatment of cells with the reducing agent DTT. Activated phosphorylated PERK is indicated by p, and the inactive form by o. Two independent experiments gave similar results. d BiP displacement from IRE1α occurs before XBP1 splicing in Flag-IRE1α-expressing IRE1α-KO MEFs infected with AdV-C5 (MOI 300). Cells were lysed and BiP–IRE1α complexes immunoprecipitated (IP) with anti-Flag antibody, and a western blot with anti-IRE1α, anti-BiP, and anti-β-tubulin antibodies was performed. A separate non-reducing immunoblot probed with anti-19K Tw1.3 antibodies revealed monomeric and dimer forms of 19K indicated as mo and di, respectively. Input lysates were 1% of the immunoprecipitated samples. The corresponding samples were also analyzed for XBP1 splicing and E1A mRNA levels by rt-PCR (reverse transcription polymerase chain reaction), as indicated. Three independent experiments gave similar results. Source data are provided as a Source Data file. e BiP–IRE1α dissociation requires E1, not 19K. Co-immunoprecipitation of Flag-IRE1α and BiP was performed as described in d with AdV mutants lacking E1 (AdV-C5-∆E1) and 19K (AdV-C5-Δ19K) at MOI 300 each. Two independent experiments gave similar results. Source data are provided as a Source Data file. f AdV-C5 infection does not increase BiP/Grp78 and IRE1α levels given in arbitrary units (a.u.), unlike the canonical UPR triggered by DTT (2 mM). The bar graph shows the normalized levels of IRE1α and BiP from three independent experiments. Data show the means ± SD from three independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Infection, Expressing, Immunofluorescence, SDS Page, Immunoprecipitation, Western Blot, Reverse Transcription Polymerase Chain Reaction

    The lumenal domain of the 19K glycoprotein activates IRE1α. a Schematic drawing showing the deletions in the E3 region of AdV mutants with yellow boxes indicating the deletions (left panel). Infection was carried out with a median of 150 particles bound per cell. Asterisk denotes a background product. At least three independent experiments gave similar results. Source data are provided as a Source Data file. b AdV-C5 19K enhances IRE1α phosphorylation, XBP1s splicing, and E1A levels. Phosphorylation of IRE1α in AdV-C5- and AdV-C5-d19K-infected HDF-TERT cells (MOI 75000, 24 hpi) was analyzed in lysates treated with or without alkaline phosphatase, and fractionated by SDS-PAGE (6%, containing 25 µM Phos-tag, first panel). XBP1 splicing in AdV-C5- and AdV-C5-d19K-infected HDF-TERT cells 24 hpi (MOI 75,000, second panel). Immunofluorescence data of E1A are shown as a scatterplot using n = 14,000 cells randomly chosen per condition, 24 hpi (MOI 75000). Central line of the box plot indicates median with first and third quartiles, and whiskers are shown as boxes and lines, respectively. Statistics were performed using the Wilcoxon two-sided nonparametric test with * p
    Figure Legend Snippet: The lumenal domain of the 19K glycoprotein activates IRE1α. a Schematic drawing showing the deletions in the E3 region of AdV mutants with yellow boxes indicating the deletions (left panel). Infection was carried out with a median of 150 particles bound per cell. Asterisk denotes a background product. At least three independent experiments gave similar results. Source data are provided as a Source Data file. b AdV-C5 19K enhances IRE1α phosphorylation, XBP1s splicing, and E1A levels. Phosphorylation of IRE1α in AdV-C5- and AdV-C5-d19K-infected HDF-TERT cells (MOI 75000, 24 hpi) was analyzed in lysates treated with or without alkaline phosphatase, and fractionated by SDS-PAGE (6%, containing 25 µM Phos-tag, first panel). XBP1 splicing in AdV-C5- and AdV-C5-d19K-infected HDF-TERT cells 24 hpi (MOI 75,000, second panel). Immunofluorescence data of E1A are shown as a scatterplot using n = 14,000 cells randomly chosen per condition, 24 hpi (MOI 75000). Central line of the box plot indicates median with first and third quartiles, and whiskers are shown as boxes and lines, respectively. Statistics were performed using the Wilcoxon two-sided nonparametric test with * p

    Techniques Used: Infection, SDS Page, Immunofluorescence

    2) Product Images from "The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections"

    Article Title: The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15844-2

    IRE1α and XBP1s facilitate persistent and lytic AdV infections. a Reduction in late AdV-C5 protein VI expression (MOI 180) of HDF-TERT cells upon RNA interference against IRE1α and XBP1 in the presence or absence of IFN-γ, including nontargeting siRNA (siNeg1). Data show the means ± SD from three independent experiments ( n = 3). b E1A expression of AdV-C5-infected HDF-TERT cells (MOI 200, 37 °C, 1 h) with or without 500 IU IFN-γ, or IRE1α RNase inhibitor 4µ8C (100 µM) at 13 days pi was analyzed by immunofluorescence showing representative images (left) and a scatterplot with 15,000 cells per condition (middle). Data show the median, first and third quartiles, and whiskers as boxes and lines, respectively. Significance was assessed with two-tailed Wilcoxon nonparametric test (middle panels). Virus titers (q-PCR, right panel) were determined after 5 days of incubation with the drug (18 d pi). Q-PCR data show the means from two technical replicates. Two independent experiments gave similar results. Scale bar, 100 µm. c xCELLigence impedance plots showing HDF-TERT cell viability upon dl309 or dl309-63/195 and AdV-C5 or AdV-C5-XBP1s-mut infections (MOI 200). Cells were seeded on xCELLigence E-16 plate, and impedance readout for cell viability was measured live at 15-min intervals. Data show the means ± SD from three technical replicates. Two independent experiments gave similar results (left row). Experimental conditions and virus amount were as in panel b Genome copy numbers of virions released to the supernatant from the same experiment. Data show the means from two technical replicates. Two independent experiments gave similar results (middle row). Representative phase-contrast images of parallel samples imaged live for dl309 or dl309-63/195 infections (images on the right side, scale bar, 200 µm). d Schematic model depicting AdV infection under the control of a five-component feedforward loop. (1) The immediate early E1A protein transactivates early promoters. including the E3 and E4, giving rise to the 19K glycoprotein (2). Activation of IRE1α by 19 K increases XBP1s mRNA, and XBP1s protein (3), which translocates into the nucleus, and binds to the E1A enhancer/promoter (e/p) of the episomal viral genome (4). Binding of XBP1s to the E1A-e/p increases the E1A levels (5), which enhances output from the E3 promoter, enhances the 19K levels, and maintains a feedforward loop supporting viral persistence and lytic infection.
    Figure Legend Snippet: IRE1α and XBP1s facilitate persistent and lytic AdV infections. a Reduction in late AdV-C5 protein VI expression (MOI 180) of HDF-TERT cells upon RNA interference against IRE1α and XBP1 in the presence or absence of IFN-γ, including nontargeting siRNA (siNeg1). Data show the means ± SD from three independent experiments ( n = 3). b E1A expression of AdV-C5-infected HDF-TERT cells (MOI 200, 37 °C, 1 h) with or without 500 IU IFN-γ, or IRE1α RNase inhibitor 4µ8C (100 µM) at 13 days pi was analyzed by immunofluorescence showing representative images (left) and a scatterplot with 15,000 cells per condition (middle). Data show the median, first and third quartiles, and whiskers as boxes and lines, respectively. Significance was assessed with two-tailed Wilcoxon nonparametric test (middle panels). Virus titers (q-PCR, right panel) were determined after 5 days of incubation with the drug (18 d pi). Q-PCR data show the means from two technical replicates. Two independent experiments gave similar results. Scale bar, 100 µm. c xCELLigence impedance plots showing HDF-TERT cell viability upon dl309 or dl309-63/195 and AdV-C5 or AdV-C5-XBP1s-mut infections (MOI 200). Cells were seeded on xCELLigence E-16 plate, and impedance readout for cell viability was measured live at 15-min intervals. Data show the means ± SD from three technical replicates. Two independent experiments gave similar results (left row). Experimental conditions and virus amount were as in panel b Genome copy numbers of virions released to the supernatant from the same experiment. Data show the means from two technical replicates. Two independent experiments gave similar results (middle row). Representative phase-contrast images of parallel samples imaged live for dl309 or dl309-63/195 infections (images on the right side, scale bar, 200 µm). d Schematic model depicting AdV infection under the control of a five-component feedforward loop. (1) The immediate early E1A protein transactivates early promoters. including the E3 and E4, giving rise to the 19K glycoprotein (2). Activation of IRE1α by 19 K increases XBP1s mRNA, and XBP1s protein (3), which translocates into the nucleus, and binds to the E1A enhancer/promoter (e/p) of the episomal viral genome (4). Binding of XBP1s to the E1A-e/p increases the E1A levels (5), which enhances output from the E3 promoter, enhances the 19K levels, and maintains a feedforward loop supporting viral persistence and lytic infection.

    Techniques Used: Expressing, Infection, Immunofluorescence, Two Tailed Test, Polymerase Chain Reaction, Incubation, Activation Assay, Binding Assay

    AdV infection of mouse embryonic fibroblasts induces XBP1 splicing and displaces BiP/Grp78 from IRE1α. a Flag-IRE1α-expressing MEFs but not IRE1α-KO MEFs induce XBP1s upon AdV-C2 infection (MOI 300) or thapsigargin (Tg, 10 µM) treatment (left panel). Representative immunofluorescence images with the anti-19K antibody 3A9 showing 19K expression in AdV-C-infected IRE1α-KO MEFs and Flag-IRE1α-expressing IRE1α-KO MEFs 24 hpi (clones 30 and 574, respectively, right panel, scale bar, 20 µm). Three independent experiments gave similar results. Source data are provided as a Source Data file. b AdV induces phosphorylation of IRE1α. Flag-IRE1α expressing IRE1α-KO MEFs at 7, 16, and 24 hpi with AdV-C5 (MOI 300) immunoblotted with anti-IRE1α antibody. Lysates were resolved on a 6% SDS-PAGE gel containing 25 µM Phos-tag. Samples were treated with or without alkaline phosphatase. Phosphorylated (p) and hypophosphorylated (o) forms of IRE1α are indicated by the dashed lines, and the percentage of IRE1α phosphorylated was calculated as indicated. Lysates are the same as in panel D demonstrating β-tubulin loading. Three independent experiments gave similar results. Source data are provided as a Source Data file. c AdV-C5 infection of HeLa cells (MOI 200) does not activate PERK, unlike treatment of cells with the reducing agent DTT. Activated phosphorylated PERK is indicated by p, and the inactive form by o. Two independent experiments gave similar results. d BiP displacement from IRE1α occurs before XBP1 splicing in Flag-IRE1α-expressing IRE1α-KO MEFs infected with AdV-C5 (MOI 300). Cells were lysed and BiP–IRE1α complexes immunoprecipitated (IP) with anti-Flag antibody, and a western blot with anti-IRE1α, anti-BiP, and anti-β-tubulin antibodies was performed. A separate non-reducing immunoblot probed with anti-19K Tw1.3 antibodies revealed monomeric and dimer forms of 19K indicated as mo and di, respectively. Input lysates were 1% of the immunoprecipitated samples. The corresponding samples were also analyzed for XBP1 splicing and E1A mRNA levels by rt-PCR (reverse transcription polymerase chain reaction), as indicated. Three independent experiments gave similar results. Source data are provided as a Source Data file. e BiP–IRE1α dissociation requires E1, not 19K. Co-immunoprecipitation of Flag-IRE1α and BiP was performed as described in d with AdV mutants lacking E1 (AdV-C5-∆E1) and 19K (AdV-C5-Δ19K) at MOI 300 each. Two independent experiments gave similar results. Source data are provided as a Source Data file. f AdV-C5 infection does not increase BiP/Grp78 and IRE1α levels given in arbitrary units (a.u.), unlike the canonical UPR triggered by DTT (2 mM). The bar graph shows the normalized levels of IRE1α and BiP from three independent experiments. Data show the means ± SD from three independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: AdV infection of mouse embryonic fibroblasts induces XBP1 splicing and displaces BiP/Grp78 from IRE1α. a Flag-IRE1α-expressing MEFs but not IRE1α-KO MEFs induce XBP1s upon AdV-C2 infection (MOI 300) or thapsigargin (Tg, 10 µM) treatment (left panel). Representative immunofluorescence images with the anti-19K antibody 3A9 showing 19K expression in AdV-C-infected IRE1α-KO MEFs and Flag-IRE1α-expressing IRE1α-KO MEFs 24 hpi (clones 30 and 574, respectively, right panel, scale bar, 20 µm). Three independent experiments gave similar results. Source data are provided as a Source Data file. b AdV induces phosphorylation of IRE1α. Flag-IRE1α expressing IRE1α-KO MEFs at 7, 16, and 24 hpi with AdV-C5 (MOI 300) immunoblotted with anti-IRE1α antibody. Lysates were resolved on a 6% SDS-PAGE gel containing 25 µM Phos-tag. Samples were treated with or without alkaline phosphatase. Phosphorylated (p) and hypophosphorylated (o) forms of IRE1α are indicated by the dashed lines, and the percentage of IRE1α phosphorylated was calculated as indicated. Lysates are the same as in panel D demonstrating β-tubulin loading. Three independent experiments gave similar results. Source data are provided as a Source Data file. c AdV-C5 infection of HeLa cells (MOI 200) does not activate PERK, unlike treatment of cells with the reducing agent DTT. Activated phosphorylated PERK is indicated by p, and the inactive form by o. Two independent experiments gave similar results. d BiP displacement from IRE1α occurs before XBP1 splicing in Flag-IRE1α-expressing IRE1α-KO MEFs infected with AdV-C5 (MOI 300). Cells were lysed and BiP–IRE1α complexes immunoprecipitated (IP) with anti-Flag antibody, and a western blot with anti-IRE1α, anti-BiP, and anti-β-tubulin antibodies was performed. A separate non-reducing immunoblot probed with anti-19K Tw1.3 antibodies revealed monomeric and dimer forms of 19K indicated as mo and di, respectively. Input lysates were 1% of the immunoprecipitated samples. The corresponding samples were also analyzed for XBP1 splicing and E1A mRNA levels by rt-PCR (reverse transcription polymerase chain reaction), as indicated. Three independent experiments gave similar results. Source data are provided as a Source Data file. e BiP–IRE1α dissociation requires E1, not 19K. Co-immunoprecipitation of Flag-IRE1α and BiP was performed as described in d with AdV mutants lacking E1 (AdV-C5-∆E1) and 19K (AdV-C5-Δ19K) at MOI 300 each. Two independent experiments gave similar results. Source data are provided as a Source Data file. f AdV-C5 infection does not increase BiP/Grp78 and IRE1α levels given in arbitrary units (a.u.), unlike the canonical UPR triggered by DTT (2 mM). The bar graph shows the normalized levels of IRE1α and BiP from three independent experiments. Data show the means ± SD from three independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Infection, Expressing, Immunofluorescence, SDS Page, Immunoprecipitation, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Related Articles

    DNA Extraction:

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    Tube Formation Assay:

    Article Title: Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells
    Article Snippet: .. Cell populations with Sox9 mutations were screened by Heteroduplex formation assay using QuickExtract DNA Extraction Solution (QE0905T, Qiagen, Peterborough, UK) and EnGen™ Mutation Detection Kit (E3321S, New England Biolabs, Herts, UK) according to the manufacturer’s instructions. .. We tested four different crRNA and the primer sets to detect mutation within each crRNA-binding region; details are provided in Supplementary Data .

    Mutagenesis:

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    Article Title: Multimodal Analysis of STRADA Function in Brain Development
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    Article Title: MYC Controls the Epstein-Barr Virus Lytic Switch.
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    Article Title: Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells
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    Article Title: Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome
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    Article Title: Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting
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    CRISPR:

    Article Title: Multimodal Analysis of STRADA Function in Brain Development
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    Polymerase Chain Reaction:

    Article Title: Development of Gene Editing Strategies for Human β-Globin (HBB) Gene Mutations
    Article Snippet: .. T7 Endonuclease Assay Thermocycler was used to denature and anneal 5 µl of PCR product as recommended by manufacturer (EnGen™ Mutation Detection Kit, NEB, E3321S). ..

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    Article Title: Multimodal Analysis of STRADA Function in Brain Development
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    Western Blot:

    Article Title: Stanniocalcin 2 (STC2) expression promotes post-radiation survival, migration and invasion of nasopharyngeal carcinoma cells
    Article Snippet: .. Selected individual colonies (STC2-KO) were expanded, screened through Western blotting and confirmed by genomic PCR, followed by T7 endonuclease 1 digestion (NEB #E3321). .. Hypoxia and glucose starvation Cells were flushed with a gas mixture of 1% O2 , 5% CO2 , and balanced N2 in hypoxia work stations (3131, Thermo scientific, USA).

    Plasmid Preparation:

    Article Title: Multimodal Analysis of STRADA Function in Brain Development
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    Cleavage Assay:

    Article Title: Generation of tryptophan hydroxylase 2 gene knockout pigs by CRISPR/Cas9-mediated gene targeting
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