Transcription factors with binding sites over-represented in promoters of genes upregulated by K13 in BCBL1 and HUVECs.

<p>(Genes upregulated more than 1.5-fold were analyzed using the JASPAR and TRANSFEC databases. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037498#s3" target="_blank">Results</a> with a p-value of less than 0.05 are shown. % input refers to the number of gene promoters bearing the specific motif compared to total number screened.</p

Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat-1

<p><b>Copyright information:</b></p><p>Taken from "Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat"</p><p>Retrovirology 2005;2():9-9.</p><p>Published online 15 Feb 2005</p><p>PMCID:PMC554086.</p><p>Copyright © 2005 Sun et al; licensee BioMed Central Ltd.</p>(75 ng/well) and an pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well) and luciferase reporter assay performed as described in Fig. 1A. The values shown are averages (mean ± SEM) of one representative experiment out of three in which each transfection was performed in duplicate. B. HIV-1 LTR activation by wild-type and mutant K13 constructs. The experiment was performed as described for Fig. 1A

Gene set enrichment analysis.

<p>For Gene set enrichment analysis of signatures genes from BCBL1-K13 (top panel) and HUVEC-K13 (lower panel), the t-test was graphed for each correlated gene in the ranked dataset. Three GSEA enrichment plots for representative biological pathways (Cytokine, NF-κB and Inflammatory) enriched in 4OHT-treated BCBL1-K13-ER^TAM and HUVEC-K13-ER^TAM are shown. The top portion of each GSEA plot shows the running enrichment score for validated genes specific for particular pathway as it moves down the ranked list. The bottom portion of each plot shows the value of ranking matrices as it moves down the list of ranked genes. The red horizontal bar which terminate with blue color indicate shift from positively correlated genes (red) to negatively correlated genes (blue). Further detailed interpretation about these plots can be found at Broad Institute web site (<a href="http://www.broadinstitute.org/gsea/index.jsp" target="_blank">http://www.broadinstitute.org/gsea/index.jsp</a>).</p

Validation of gene array data by qRT-PCR.

<p>(A) Twenty five genes from NF-κB, cytokine, and inflammatory pathways were randomly selected and their relative mRNA levels in mock and 4OHT-treated vector and K13-ER^TAM-expressing BCBL1 cells were examined using qRT-PCR. Real-time PCR reactions were performed in triplicate and the data presented as fold change mean ±S.E in target gene expression (*p<0.05; Student's t-test). (B) Pearson Correlation coefficient between gene expression array and real time PCR showed a significant agreement (Correlation coefficient 0.88; p<0.0001).</p

NF-κB inhibitors block K13-regulated genes.

<p>BCBL1 K13-ER^TAM cells were treated with two NF–κB inhibitors (2 µM Bay 11-7082 or 2 µM As2O3) for 2 hours followed by 4OHT treatment for additional 24 hours and total RNA was isolated as described in the Materials and Method section. Nine genes were randomly picked and their relative mRNA levels were examined using real-time RT-PCR as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037498#pone-0037498-g002" target="_blank">Figure 2A</a>.</p

Gene sets enriched in BCBL-K13 and HUVEC-K13 cells (a) Biocarta gene sets, (b) KEGG (Kyoto Encyclopedia of Genes and Genomes) gene sets.

<p>Gene sets enriched in BCBL-K13 and HUVEC-K13 cells (a) Biocarta gene sets, (b) KEGG (Kyoto Encyclopedia of Genes and Genomes) gene sets.</p

Summary of differentially regulated gene clusters in 4OHT-treated K13-ER^TAM-transduced BCBL1 cells.

<p>Summary of differentially regulated gene clusters in 4OHT-treated K13-ER^TAM-transduced BCBL1 cells.</p

Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat-0

<p><b>Copyright information:</b></p><p>Taken from "Kaposi's sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1 Long Terminal Repeat via the classical NF-κB pathway and functionally cooperates with Tat"</p><p>Retrovirology 2005;2():9-9.</p><p>Published online 15 Feb 2005</p><p>PMCID:PMC554086.</p><p>Copyright © 2005 Sun et al; licensee BioMed Central Ltd.</p>onstruct (75 ng/well), and the experiment was performed as described under "Materials and Methods." The values shown are averages (Mean ± S.E.) of one representative experiment out of three in which each transfection was performed in duplicate. B. A dose-response analysis of HIV-1 LTR activation by K13 and pro-inflammatory cytokines. 293T cells were transfected with the indicated amounts of a K13 expression plasmid and luciferase assay performed 36 h post-transfection as described for (A). The total amount of transfected DNA was kept constant by adding an empty vector. For experiments involving TNF-α and IL-1β, cells were treated with the indicated concentration of cytokines 12 h after transfection of the reporter plasmids and assayed for reporter activity after 24 h of stimulation. C. K13 activates HIV-1 LTR in Cos-7 cells. The experiment was performed as described in 1A except LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA) was used for transfection and Renilla luciferase was used for normalization. D. K13 activates HIV-1 LTR in Jurkat cells. The experiment was performed as described for 1C by using LIPOFECTAMINE 2000 Reagent (Invitrogen, Carlsbad, CA)

HOIL-1 is not essential for K13-induced NF-κB activation.

<p><b>A.</b> The expression of FLAG-tagged K13-ER^TAM in wild-type and <i>HOIL-1^−/−</i> MEF was confirmed with Western blotting. The blot was re-probed with a tubulin antibody to show equal protein loading. <b>B.</b> Wild-type and <i>HOIL-1^−/−</i> MEFs stably expressing FLAG-K13-ER^TAM were transfected with NF-κB-Luc and Renilla reporter constructs. Cells were subsequently treated with 4OHT (20 nM) for 48 hours and the luciferase reporter assay was performed essentially as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#pone-0036601-g001" target="_blank">Figure 1A</a>. Asterisks (*) indicate significance at levels of p≤0.05 as compared to vehicle-treated cells. <b>C.</b> Wild-type and <i>HOIL-1^−/−</i> MEFs were transfected with NF-κB-Luc and Renilla reporter constructs and 6 hours post-transfection, these cells were treated with mTNF-α (10ng/ml) for 18 hours and the luciferase reporter assay was performed essentially as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#pone-0036601-g001" target="_blank">Figure 1A</a>. <b>D.</b> Expression of transduced FLAG-tagged K13 in wild-type and <i>HOIL-1^−/−</i> MEFs was examined by immunoblot analysis; tubulin was used as a loading control. <b>E.</b> Nuclear p65 DNA binding activities in the nuclear extracts of wild-type and <i>HOIL-1^−/−</i> MEFs expressing an empty vector or FLAG-K13. Asterisks (*) indicate significance at levels of p≤0.05 as compared to vector cells. <b>F.</b> Nuclear p65 DNA binding activities in the nuclear extracts of wild-type and <i>HOIL-1^−/−</i> MEFs following treatment with murine TNFα. <b>G.</b> Wild-type and <i>HOIL-1^−/−</i> MEFs expressing FLAG-K13 were examined for NF-κB activation by Western blot analysis using antibodies against phospho-IκBα, Total IκBα, A20 and RelB. The blot was re-probed with FLAG and Tubulin antibodies to check the expression of the transduced K13 and equal protein loading, respectively.</p

K13 activates NF-κB pathway without involving TAK1. A.

<p>Immunoblot showing expression of ectopically expressed FLAG-tagged K13 in <i>TAK1^+/+</i> and <i>TAK1^−/−</i> MEFs and endogenous K13 in BC1 cell line. <b>B.</b> Status of NF-κB pathway, as measured by an EMSA in <i>TAK1^+/+</i> and <i>TAK1^−/−</i> MEF cells stably expressing vector and K13. The position of the induced NF-κB complexes is marked by an <i>asterisk</i>, while an <i>arrow</i> marks the position of the constitutive complexes. <b>C.</b> The NF-κB subunits composition of the K13-induced nuclear NF-κB complexes in the wild-type and <i>TAK1^−/−</i> MEFs was determined with an ELISA-based DNA-binding assay performed in triplicate. Asterisks (*) indicate significance at levels of p≤0.05. <b>D. </b><i>TAK1^+/+</i> and <i>TAK1^−/−</i> MEFs expressing FLAG-K13-ER^TAM were transfected with NF-κB-Luc and Renilla reporter constructs and subsequently treated with 4OHT (20 nM) for 48 hours and the reporter assays performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036601#pone-0036601-g001" target="_blank">Figure 1A</a>. Asterisks (*) indicate significance at levels of p≤0.05. <b>E.</b> Nuclear extracts from Jurkat, BC1, BCBL1 and Namalwa were used for the measurement of p65 DNA-binding activity using an ELISA-based assay. <b>F.</b> Lack of phosphorylation of TAK1 by K13. Cell lysates prepared from BCBL1 and Namalwa cells expressing an empty vector or FLAG-K13 were probed with an antibody to detect phosphorylation of TAK1. Vector cells treated with TNFα were used a positive control. The blot was re-probed with FLAG and Tubulin antibodies to check the expression of the transduced K13 and equal protein loading, respectively. <b>G</b>−<b>H.</b> Treatment with TAK1 inhibitor 5Z-7-oxo-zeaenol had no effect on 4OHT induced NF-κB-Luc activity in 293-K13-ER^TAM-NF-κB-Luc cells but effectively blocked TNFα and IL1β induced NF-κB-Luc activity.</p