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orf21 probe number acd 559011  (Advanced Cell Diagnostics Inc)

 
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    Advanced Cell Diagnostics Inc orf21 probe number acd 559011
    Schematic of <t>ORF21</t> luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).
    Orf21 Probe Number Acd 559011, supplied by Advanced Cell Diagnostics Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/orf21+probe+number+acd+559011/pmc07022350-452-0-5?v=Advanced+Cell+Diagnostics+Inc
    Average 90 stars, based on 1 article reviews
    orf21 probe number acd 559011 - by Bioz Stars, 2026-07
    90/100 stars

    Images

    1) Product Images from "Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1"

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.01555-19

    Schematic of ORF21 luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).
    Figure Legend Snippet: Schematic of ORF21 luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).

    Techniques Used: Luciferase, Construct, Activation Assay, Comparison, Control, Plasmid Preparation, Expressing, Transfection

    Effect of XRE3 and XRE4 mutations in the ORF21 promoter on the response to XBP1s. (A) Construct of the wild-type ORF21 and mutant reporter plasmids. Three different mutant reporters for each XRE were constructed in the pORF21-1239 full-length promoter, containing a 2- to 4-bp substitution within core XRE sequences. DNA sequences for the XRE3, XRE4 wild type, and mutant plasmids are shown. The underlined regions and bold letters indicate the mutations from wild type for each mutant construct. (B) Comparison of the activation of wild-type pORF21-1239 luciferase reporter with the XRE3 or XRE4 mutant luciferase reporters by XBP-1s. HEK-293T cells were cotransfected with 300 ng of pORF21-1239WT or X3 or X4 M1, 2, or 3 promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 expression plasmid control (gray bars). Values are expressed as fold increase over the pGL3basic reporter transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations. **P < 0.01 and ***P < 0.005 for the comparisons shown; none of the comparisons between the X4 mutations and wild type (WT) were significant (P > 0.05).
    Figure Legend Snippet: Effect of XRE3 and XRE4 mutations in the ORF21 promoter on the response to XBP1s. (A) Construct of the wild-type ORF21 and mutant reporter plasmids. Three different mutant reporters for each XRE were constructed in the pORF21-1239 full-length promoter, containing a 2- to 4-bp substitution within core XRE sequences. DNA sequences for the XRE3, XRE4 wild type, and mutant plasmids are shown. The underlined regions and bold letters indicate the mutations from wild type for each mutant construct. (B) Comparison of the activation of wild-type pORF21-1239 luciferase reporter with the XRE3 or XRE4 mutant luciferase reporters by XBP-1s. HEK-293T cells were cotransfected with 300 ng of pORF21-1239WT or X3 or X4 M1, 2, or 3 promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 expression plasmid control (gray bars). Values are expressed as fold increase over the pGL3basic reporter transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations. **P < 0.01 and ***P < 0.005 for the comparisons shown; none of the comparisons between the X4 mutations and wild type (WT) were significant (P > 0.05).

    Techniques Used: Construct, Mutagenesis, Comparison, Activation Assay, Luciferase, Control, Plasmid Preparation, Expressing, Transfection

    The ORF21 promoter contains potential HREs but does not respond to hypoxia in the absence of XBP-1s. (A). Comparison of the activation of wild-type pORF21 and pORF36 luciferase reporter in response to hypoxia. HEK-293T cells were cotransfected with 300 ng of pORF21 or 36 luciferase promoter, 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 control (gray bars), and 50 ng of an internal β-Gal control plasmid, cultured in normoxia for 32 hours, and then cells were treated in normoxic or hypoxic (1% oxygen) conditions for 16 hours. Values are expressed as the fold increase over the value for the pGL3 basic reporter transfected with an empty expression vector (pcDNA3.1) in normoxia and represent the mean of three independent experiments. Error bars denote the standard deviations. (*P ≤ 0.05; NS, not significant). (B). Comparison of the activation of the wild-type pORF21-1239 luciferase reporter or the HIF-responsive pORF36 reporter in response to degradation-resistant HIF-1 (dr-HIF-1). HEK-293T cells were cotransfected with 300 ng of the pORF21-1239 luciferase reporter or pORF36 promoter and 50 ng of an internal-β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding dr-HIF-1 or the pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value of the pGL3basic reporter transfected with an empty expression vector pcDNA3.1 for each reporter construct and represent the mean of three independent experiments. Error bars denote standard deviation; ****P ≤ 0.001; NS, not significant. (C) Western blot showing XBP-1s (55 kDa) and RTA (85 kDa) expression in BCBL-1 cells 48 hours after treatment with 0.5 μg/ml to 2.5 μg/ml of tunicamycin. As seen, TM induces XBP-1s and RTA but does not induce HIF-1α production in BCBL-1 cells. However, HIF-1α protein expression is seen in BCBCl-1 cells 48 hours after treatment with CoCl2 at 75 μM. Actin was used as the loading control, and TPA was a control for KSHV activation. (D) Comparison of the activation of the pORF21-1239 luciferase reporter by RTA, XBP-1s, or both. HEK-293T cells were cotransfected with 300 ng of pGL3b control reporter plasmid DNA or the pORF21-1239 promoter luciferase reporter and 50 ng of an internal β-Gal control plasmid in the presence of 10 ng of a DNA expression plasmid encoding RTA and 100 ng pcDNA3.1, 100 ng of an expression plasmid for XBP-1s and 10 ng pcDNA3.1, or both RTA and XBP-1s expression plasmids. Values are expressed as the fold increase over the value for the pGL3b basic reporter transfected with pcDNA3.1. Shown are the mean ± standard deviation of triplicate determinations from one representative experiment expressed as the fold change compared with the level of nontreated cells.
    Figure Legend Snippet: The ORF21 promoter contains potential HREs but does not respond to hypoxia in the absence of XBP-1s. (A). Comparison of the activation of wild-type pORF21 and pORF36 luciferase reporter in response to hypoxia. HEK-293T cells were cotransfected with 300 ng of pORF21 or 36 luciferase promoter, 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 control (gray bars), and 50 ng of an internal β-Gal control plasmid, cultured in normoxia for 32 hours, and then cells were treated in normoxic or hypoxic (1% oxygen) conditions for 16 hours. Values are expressed as the fold increase over the value for the pGL3 basic reporter transfected with an empty expression vector (pcDNA3.1) in normoxia and represent the mean of three independent experiments. Error bars denote the standard deviations. (*P ≤ 0.05; NS, not significant). (B). Comparison of the activation of the wild-type pORF21-1239 luciferase reporter or the HIF-responsive pORF36 reporter in response to degradation-resistant HIF-1 (dr-HIF-1). HEK-293T cells were cotransfected with 300 ng of the pORF21-1239 luciferase reporter or pORF36 promoter and 50 ng of an internal-β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding dr-HIF-1 or the pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value of the pGL3basic reporter transfected with an empty expression vector pcDNA3.1 for each reporter construct and represent the mean of three independent experiments. Error bars denote standard deviation; ****P ≤ 0.001; NS, not significant. (C) Western blot showing XBP-1s (55 kDa) and RTA (85 kDa) expression in BCBL-1 cells 48 hours after treatment with 0.5 μg/ml to 2.5 μg/ml of tunicamycin. As seen, TM induces XBP-1s and RTA but does not induce HIF-1α production in BCBL-1 cells. However, HIF-1α protein expression is seen in BCBCl-1 cells 48 hours after treatment with CoCl2 at 75 μM. Actin was used as the loading control, and TPA was a control for KSHV activation. (D) Comparison of the activation of the pORF21-1239 luciferase reporter by RTA, XBP-1s, or both. HEK-293T cells were cotransfected with 300 ng of pGL3b control reporter plasmid DNA or the pORF21-1239 promoter luciferase reporter and 50 ng of an internal β-Gal control plasmid in the presence of 10 ng of a DNA expression plasmid encoding RTA and 100 ng pcDNA3.1, 100 ng of an expression plasmid for XBP-1s and 10 ng pcDNA3.1, or both RTA and XBP-1s expression plasmids. Values are expressed as the fold increase over the value for the pGL3b basic reporter transfected with pcDNA3.1. Shown are the mean ± standard deviation of triplicate determinations from one representative experiment expressed as the fold change compared with the level of nontreated cells.

    Techniques Used: Comparison, Activation Assay, Luciferase, Expressing, Plasmid Preparation, Control, Cell Culture, Transfection, Construct, Standard Deviation, Western Blot

    ChIP showing binding of XBP-1s to the ORF21 promoter in TM-treated cells. BCBL-1 cells were treated with DMSO (A) or TM treatment (0.5 μg/ml) (B) for 48 hours to induce XBP1s and then cross-linked. Chromatin IP of fragmented DNA was performed with anti-XBP-1 antibody, CHIP positive-control anti-histone H3 antibody, or control IgG. Precipitated DNA was assayed by qPCR with specific primers for amplification of XRE2, 3, or 4 of the ORF21 promoter and with primers for an ORF21, ORF36 non-XRE region as a negative control. The data were quantitated as described in the Materials and Methods. Results shown are the mean ± standard deviation of triplicate determinations from a typical experiment of three experiments performed. In controls performed at the same time, DNA immunoprecipitated with histone H3 antibody, but not XBP-1 antibody or control IgG, was enriched for RPL30 exon 3.
    Figure Legend Snippet: ChIP showing binding of XBP-1s to the ORF21 promoter in TM-treated cells. BCBL-1 cells were treated with DMSO (A) or TM treatment (0.5 μg/ml) (B) for 48 hours to induce XBP1s and then cross-linked. Chromatin IP of fragmented DNA was performed with anti-XBP-1 antibody, CHIP positive-control anti-histone H3 antibody, or control IgG. Precipitated DNA was assayed by qPCR with specific primers for amplification of XRE2, 3, or 4 of the ORF21 promoter and with primers for an ORF21, ORF36 non-XRE region as a negative control. The data were quantitated as described in the Materials and Methods. Results shown are the mean ± standard deviation of triplicate determinations from a typical experiment of three experiments performed. In controls performed at the same time, DNA immunoprecipitated with histone H3 antibody, but not XBP-1 antibody or control IgG, was enriched for RPL30 exon 3.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Positive Control, Control, Amplification, Negative Control, Standard Deviation, Immunoprecipitation

    XBP-1, ORF21, RTA ,and vIL-6 mRNA upregulation mediated by TM, a chemical inducer of XBP-1s, in the BCBL-1 PEL line. BCBL-1 cells were treated with increasing doses of TM to induce ER stress; cells were also treated with TPA as an inducer of RTA or a DMSO control. Real-time quantitative PCR showing expression of spliced XBP-1 (A) and total XBP-1 (B) in BCBL-1 cells treated with the compounds shown for 4 h, 8 h, and 24 h. (C, D, and E) Real-time quantitative PCR showing expression of ORF21, vIL-6, and RTA mRNA in BCBL-1 cells cultured in the same way and harvested at 4 hours (C), 8 hours (D), and 24 hours (E). Shown are the mean ± the standard deviation of triplicate determinations from one representative experiment out of three expressed as the fold change compared with the DMSO control.
    Figure Legend Snippet: XBP-1, ORF21, RTA ,and vIL-6 mRNA upregulation mediated by TM, a chemical inducer of XBP-1s, in the BCBL-1 PEL line. BCBL-1 cells were treated with increasing doses of TM to induce ER stress; cells were also treated with TPA as an inducer of RTA or a DMSO control. Real-time quantitative PCR showing expression of spliced XBP-1 (A) and total XBP-1 (B) in BCBL-1 cells treated with the compounds shown for 4 h, 8 h, and 24 h. (C, D, and E) Real-time quantitative PCR showing expression of ORF21, vIL-6, and RTA mRNA in BCBL-1 cells cultured in the same way and harvested at 4 hours (C), 8 hours (D), and 24 hours (E). Shown are the mean ± the standard deviation of triplicate determinations from one representative experiment out of three expressed as the fold change compared with the DMSO control.

    Techniques Used: Control, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Standard Deviation

    RNAscope analysis of ORF21 and XBP-1 in representative sections of a lymph node from a patient with KSHV-MCD. (A and B) A paraformaldehyde-fixed paraffin-embedded lymph node from a patient with KSHV-MCD was analyzed for ORF21 (green) and XBP-1 (red) mRNA as described in the Materials and Methods. The white arrows denote cells that express both ORF21 and XBP-1, while the pink arrow denotes a cell that expresses ORF21 only. In addition, CD20 protein expression is identified by immunohistofluorescence (blue), and nuclei identified by 4′,6-diamidino-2-phenylindole (DAPI) is shown in gray. (C) A probe-control section stained for CD20. It is worth noting that most KSHV plasmablasts do not express CD20. The scale bar is 100 μm.
    Figure Legend Snippet: RNAscope analysis of ORF21 and XBP-1 in representative sections of a lymph node from a patient with KSHV-MCD. (A and B) A paraformaldehyde-fixed paraffin-embedded lymph node from a patient with KSHV-MCD was analyzed for ORF21 (green) and XBP-1 (red) mRNA as described in the Materials and Methods. The white arrows denote cells that express both ORF21 and XBP-1, while the pink arrow denotes a cell that expresses ORF21 only. In addition, CD20 protein expression is identified by immunohistofluorescence (blue), and nuclei identified by 4′,6-diamidino-2-phenylindole (DAPI) is shown in gray. (C) A probe-control section stained for CD20. It is worth noting that most KSHV plasmablasts do not express CD20. The scale bar is 100 μm.

    Techniques Used: RNAscope, Expressing, Immunohistofluorescence, Control, Staining

    Primers used in real-time PCR
    Figure Legend Snippet: Primers used in real-time PCR

    Techniques Used: Sequencing



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    orf21 probe number acd 559011  (Advanced Cell Diagnostics Inc)
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    Advanced Cell Diagnostics Inc orf21 probe number acd 559011
    Schematic of <t>ORF21</t> luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).
    Orf21 Probe Number Acd 559011, supplied by Advanced Cell Diagnostics Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/orf21+probe+number+acd+559011/pmc07022350-452-0-5?v=Advanced+Cell+Diagnostics+Inc
    Average 90 stars, based on 1 article reviews
    orf21 probe number acd 559011 - by Bioz Stars, 2026-07
    90/100 stars
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    Schematic of ORF21 luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).

    Journal: Journal of Virology

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    doi: 10.1128/JVI.01555-19

    Figure Lengend Snippet: Schematic of ORF21 luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).

    Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

    Techniques: Luciferase, Construct, Activation Assay, Comparison, Control, Plasmid Preparation, Expressing, Transfection

    Effect of XRE3 and XRE4 mutations in the ORF21 promoter on the response to XBP1s. (A) Construct of the wild-type ORF21 and mutant reporter plasmids. Three different mutant reporters for each XRE were constructed in the pORF21-1239 full-length promoter, containing a 2- to 4-bp substitution within core XRE sequences. DNA sequences for the XRE3, XRE4 wild type, and mutant plasmids are shown. The underlined regions and bold letters indicate the mutations from wild type for each mutant construct. (B) Comparison of the activation of wild-type pORF21-1239 luciferase reporter with the XRE3 or XRE4 mutant luciferase reporters by XBP-1s. HEK-293T cells were cotransfected with 300 ng of pORF21-1239WT or X3 or X4 M1, 2, or 3 promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 expression plasmid control (gray bars). Values are expressed as fold increase over the pGL3basic reporter transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations. **P < 0.01 and ***P < 0.005 for the comparisons shown; none of the comparisons between the X4 mutations and wild type (WT) were significant (P > 0.05).

    Journal: Journal of Virology

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    doi: 10.1128/JVI.01555-19

    Figure Lengend Snippet: Effect of XRE3 and XRE4 mutations in the ORF21 promoter on the response to XBP1s. (A) Construct of the wild-type ORF21 and mutant reporter plasmids. Three different mutant reporters for each XRE were constructed in the pORF21-1239 full-length promoter, containing a 2- to 4-bp substitution within core XRE sequences. DNA sequences for the XRE3, XRE4 wild type, and mutant plasmids are shown. The underlined regions and bold letters indicate the mutations from wild type for each mutant construct. (B) Comparison of the activation of wild-type pORF21-1239 luciferase reporter with the XRE3 or XRE4 mutant luciferase reporters by XBP-1s. HEK-293T cells were cotransfected with 300 ng of pORF21-1239WT or X3 or X4 M1, 2, or 3 promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 expression plasmid control (gray bars). Values are expressed as fold increase over the pGL3basic reporter transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations. **P < 0.01 and ***P < 0.005 for the comparisons shown; none of the comparisons between the X4 mutations and wild type (WT) were significant (P > 0.05).

    Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

    Techniques: Construct, Mutagenesis, Comparison, Activation Assay, Luciferase, Control, Plasmid Preparation, Expressing, Transfection

    The ORF21 promoter contains potential HREs but does not respond to hypoxia in the absence of XBP-1s. (A). Comparison of the activation of wild-type pORF21 and pORF36 luciferase reporter in response to hypoxia. HEK-293T cells were cotransfected with 300 ng of pORF21 or 36 luciferase promoter, 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 control (gray bars), and 50 ng of an internal β-Gal control plasmid, cultured in normoxia for 32 hours, and then cells were treated in normoxic or hypoxic (1% oxygen) conditions for 16 hours. Values are expressed as the fold increase over the value for the pGL3 basic reporter transfected with an empty expression vector (pcDNA3.1) in normoxia and represent the mean of three independent experiments. Error bars denote the standard deviations. (*P ≤ 0.05; NS, not significant). (B). Comparison of the activation of the wild-type pORF21-1239 luciferase reporter or the HIF-responsive pORF36 reporter in response to degradation-resistant HIF-1 (dr-HIF-1). HEK-293T cells were cotransfected with 300 ng of the pORF21-1239 luciferase reporter or pORF36 promoter and 50 ng of an internal-β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding dr-HIF-1 or the pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value of the pGL3basic reporter transfected with an empty expression vector pcDNA3.1 for each reporter construct and represent the mean of three independent experiments. Error bars denote standard deviation; ****P ≤ 0.001; NS, not significant. (C) Western blot showing XBP-1s (55 kDa) and RTA (85 kDa) expression in BCBL-1 cells 48 hours after treatment with 0.5 μg/ml to 2.5 μg/ml of tunicamycin. As seen, TM induces XBP-1s and RTA but does not induce HIF-1α production in BCBL-1 cells. However, HIF-1α protein expression is seen in BCBCl-1 cells 48 hours after treatment with CoCl2 at 75 μM. Actin was used as the loading control, and TPA was a control for KSHV activation. (D) Comparison of the activation of the pORF21-1239 luciferase reporter by RTA, XBP-1s, or both. HEK-293T cells were cotransfected with 300 ng of pGL3b control reporter plasmid DNA or the pORF21-1239 promoter luciferase reporter and 50 ng of an internal β-Gal control plasmid in the presence of 10 ng of a DNA expression plasmid encoding RTA and 100 ng pcDNA3.1, 100 ng of an expression plasmid for XBP-1s and 10 ng pcDNA3.1, or both RTA and XBP-1s expression plasmids. Values are expressed as the fold increase over the value for the pGL3b basic reporter transfected with pcDNA3.1. Shown are the mean ± standard deviation of triplicate determinations from one representative experiment expressed as the fold change compared with the level of nontreated cells.

    Journal: Journal of Virology

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    doi: 10.1128/JVI.01555-19

    Figure Lengend Snippet: The ORF21 promoter contains potential HREs but does not respond to hypoxia in the absence of XBP-1s. (A). Comparison of the activation of wild-type pORF21 and pORF36 luciferase reporter in response to hypoxia. HEK-293T cells were cotransfected with 300 ng of pORF21 or 36 luciferase promoter, 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 control (gray bars), and 50 ng of an internal β-Gal control plasmid, cultured in normoxia for 32 hours, and then cells were treated in normoxic or hypoxic (1% oxygen) conditions for 16 hours. Values are expressed as the fold increase over the value for the pGL3 basic reporter transfected with an empty expression vector (pcDNA3.1) in normoxia and represent the mean of three independent experiments. Error bars denote the standard deviations. (*P ≤ 0.05; NS, not significant). (B). Comparison of the activation of the wild-type pORF21-1239 luciferase reporter or the HIF-responsive pORF36 reporter in response to degradation-resistant HIF-1 (dr-HIF-1). HEK-293T cells were cotransfected with 300 ng of the pORF21-1239 luciferase reporter or pORF36 promoter and 50 ng of an internal-β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding dr-HIF-1 or the pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value of the pGL3basic reporter transfected with an empty expression vector pcDNA3.1 for each reporter construct and represent the mean of three independent experiments. Error bars denote standard deviation; ****P ≤ 0.001; NS, not significant. (C) Western blot showing XBP-1s (55 kDa) and RTA (85 kDa) expression in BCBL-1 cells 48 hours after treatment with 0.5 μg/ml to 2.5 μg/ml of tunicamycin. As seen, TM induces XBP-1s and RTA but does not induce HIF-1α production in BCBL-1 cells. However, HIF-1α protein expression is seen in BCBCl-1 cells 48 hours after treatment with CoCl2 at 75 μM. Actin was used as the loading control, and TPA was a control for KSHV activation. (D) Comparison of the activation of the pORF21-1239 luciferase reporter by RTA, XBP-1s, or both. HEK-293T cells were cotransfected with 300 ng of pGL3b control reporter plasmid DNA or the pORF21-1239 promoter luciferase reporter and 50 ng of an internal β-Gal control plasmid in the presence of 10 ng of a DNA expression plasmid encoding RTA and 100 ng pcDNA3.1, 100 ng of an expression plasmid for XBP-1s and 10 ng pcDNA3.1, or both RTA and XBP-1s expression plasmids. Values are expressed as the fold increase over the value for the pGL3b basic reporter transfected with pcDNA3.1. Shown are the mean ± standard deviation of triplicate determinations from one representative experiment expressed as the fold change compared with the level of nontreated cells.

    Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

    Techniques: Comparison, Activation Assay, Luciferase, Expressing, Plasmid Preparation, Control, Cell Culture, Transfection, Construct, Standard Deviation, Western Blot

    ChIP showing binding of XBP-1s to the ORF21 promoter in TM-treated cells. BCBL-1 cells were treated with DMSO (A) or TM treatment (0.5 μg/ml) (B) for 48 hours to induce XBP1s and then cross-linked. Chromatin IP of fragmented DNA was performed with anti-XBP-1 antibody, CHIP positive-control anti-histone H3 antibody, or control IgG. Precipitated DNA was assayed by qPCR with specific primers for amplification of XRE2, 3, or 4 of the ORF21 promoter and with primers for an ORF21, ORF36 non-XRE region as a negative control. The data were quantitated as described in the Materials and Methods. Results shown are the mean ± standard deviation of triplicate determinations from a typical experiment of three experiments performed. In controls performed at the same time, DNA immunoprecipitated with histone H3 antibody, but not XBP-1 antibody or control IgG, was enriched for RPL30 exon 3.

    Journal: Journal of Virology

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    doi: 10.1128/JVI.01555-19

    Figure Lengend Snippet: ChIP showing binding of XBP-1s to the ORF21 promoter in TM-treated cells. BCBL-1 cells were treated with DMSO (A) or TM treatment (0.5 μg/ml) (B) for 48 hours to induce XBP1s and then cross-linked. Chromatin IP of fragmented DNA was performed with anti-XBP-1 antibody, CHIP positive-control anti-histone H3 antibody, or control IgG. Precipitated DNA was assayed by qPCR with specific primers for amplification of XRE2, 3, or 4 of the ORF21 promoter and with primers for an ORF21, ORF36 non-XRE region as a negative control. The data were quantitated as described in the Materials and Methods. Results shown are the mean ± standard deviation of triplicate determinations from a typical experiment of three experiments performed. In controls performed at the same time, DNA immunoprecipitated with histone H3 antibody, but not XBP-1 antibody or control IgG, was enriched for RPL30 exon 3.

    Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Positive Control, Control, Amplification, Negative Control, Standard Deviation, Immunoprecipitation

    XBP-1, ORF21, RTA ,and vIL-6 mRNA upregulation mediated by TM, a chemical inducer of XBP-1s, in the BCBL-1 PEL line. BCBL-1 cells were treated with increasing doses of TM to induce ER stress; cells were also treated with TPA as an inducer of RTA or a DMSO control. Real-time quantitative PCR showing expression of spliced XBP-1 (A) and total XBP-1 (B) in BCBL-1 cells treated with the compounds shown for 4 h, 8 h, and 24 h. (C, D, and E) Real-time quantitative PCR showing expression of ORF21, vIL-6, and RTA mRNA in BCBL-1 cells cultured in the same way and harvested at 4 hours (C), 8 hours (D), and 24 hours (E). Shown are the mean ± the standard deviation of triplicate determinations from one representative experiment out of three expressed as the fold change compared with the DMSO control.

    Journal: Journal of Virology

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    doi: 10.1128/JVI.01555-19

    Figure Lengend Snippet: XBP-1, ORF21, RTA ,and vIL-6 mRNA upregulation mediated by TM, a chemical inducer of XBP-1s, in the BCBL-1 PEL line. BCBL-1 cells were treated with increasing doses of TM to induce ER stress; cells were also treated with TPA as an inducer of RTA or a DMSO control. Real-time quantitative PCR showing expression of spliced XBP-1 (A) and total XBP-1 (B) in BCBL-1 cells treated with the compounds shown for 4 h, 8 h, and 24 h. (C, D, and E) Real-time quantitative PCR showing expression of ORF21, vIL-6, and RTA mRNA in BCBL-1 cells cultured in the same way and harvested at 4 hours (C), 8 hours (D), and 24 hours (E). Shown are the mean ± the standard deviation of triplicate determinations from one representative experiment out of three expressed as the fold change compared with the DMSO control.

    Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

    Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Standard Deviation

    RNAscope analysis of ORF21 and XBP-1 in representative sections of a lymph node from a patient with KSHV-MCD. (A and B) A paraformaldehyde-fixed paraffin-embedded lymph node from a patient with KSHV-MCD was analyzed for ORF21 (green) and XBP-1 (red) mRNA as described in the Materials and Methods. The white arrows denote cells that express both ORF21 and XBP-1, while the pink arrow denotes a cell that expresses ORF21 only. In addition, CD20 protein expression is identified by immunohistofluorescence (blue), and nuclei identified by 4′,6-diamidino-2-phenylindole (DAPI) is shown in gray. (C) A probe-control section stained for CD20. It is worth noting that most KSHV plasmablasts do not express CD20. The scale bar is 100 μm.

    Journal: Journal of Virology

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    doi: 10.1128/JVI.01555-19

    Figure Lengend Snippet: RNAscope analysis of ORF21 and XBP-1 in representative sections of a lymph node from a patient with KSHV-MCD. (A and B) A paraformaldehyde-fixed paraffin-embedded lymph node from a patient with KSHV-MCD was analyzed for ORF21 (green) and XBP-1 (red) mRNA as described in the Materials and Methods. The white arrows denote cells that express both ORF21 and XBP-1, while the pink arrow denotes a cell that expresses ORF21 only. In addition, CD20 protein expression is identified by immunohistofluorescence (blue), and nuclei identified by 4′,6-diamidino-2-phenylindole (DAPI) is shown in gray. (C) A probe-control section stained for CD20. It is worth noting that most KSHV plasmablasts do not express CD20. The scale bar is 100 μm.

    Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

    Techniques: RNAscope, Expressing, Immunohistofluorescence, Control, Staining

    Primers used in real-time PCR

    Journal: Journal of Virology

    Article Title: Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

    doi: 10.1128/JVI.01555-19

    Figure Lengend Snippet: Primers used in real-time PCR

    Article Snippet: ORF21 (probe number ACD 559011; ACD Bio, Newark, CA) and XBP-1 (probe number ACD 436251) RNA was detected using next-generation, ultrasensitive in situ hybridization technology as previously described ( 60 ).

    Techniques: Sequencing