Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) Analysis of phosphorylated (active) STAT1 and STAT2 (pSTAT1, pSTAT2) and total STAT1 and STAT2 in latent (-Dox) and lytic (+Dox, 2 days) iBCBL-1 PEL cells, transduced with lentivirus vector expressing either non-silencing control (ns) or vIRF-1 mRNA-specific (virf1) shRNA. Cultures remained untreated (mock) or were treated with IFNβ (50 ng/ml). Derived cell extracts were immunoblotted for assessments of pSTAT1 and pSTAT2 levels, in addition to validation of vIRF-1 induction (+Dox) and depletion and monitoring of IRF9 expression. Quantified pSTAT1 (pS1) and pSTAT2 (pS2) levels for IFNβ-treated samples, normalized to total STAT1 (S1) and STAT2 (S2), respectively, and expressed as ratios of levels in vIRF-1 versus control shRNA-transduced cells (virf1/ns), are shown below the blots. Dotted lines indicate rearrangement (for consistency) and deletion of lanes. (B) Analysis of STAT1 and STAT2 regulation as a function of vIRF-1 in BAC16 HHV-8-infected iSLK cells. Cultures infected with wild-type (wt) or vIRF-1-knockout (ttg) virus were treated with Dox and sodium butyrate (NaB) to induce lytic replication or left untreated (latent infection), with or without IFNβ treatment. Cells were harvested 3 days after treatment, and cell lysates were analyzed as above. Levels of pSTAT1 and pSTAT2, normalized to total STAT1 and STAT2, in ttg-virus-infected cells relative to wt-virus-infected cells are indicated below the blots. Arrowheads indicate positions of pSTAT1 and vIRF-1 bands; asterisks indicates non-specific bands. For A and B: nd, not determined; -, undetectable pSTAT. (C) Detection of lytic cycle-regulated ISG15 and ISG56 in iBCBL-1 cells. Expression of representative ISGs, ISG15 and ISG56, and also IFNβ, were assessed in latent and (1-day) lytic cells by RT-qPCR analysis of the respective transcripts. Data are from biological triplicates; error bars represent standard deviations from the means, and student t-test P values (two-tailed) are shown. (D) ISG15 and ISG56 transcript levels were assessed in lytically-infected iBCBL-1 cells 2 days post-Dox treatment, with or without vIRF-1 depletion, in the presence (IFNβ) or absence (mock) of IFNβ treatment (50 ng/ml). RT-qPCR for vIRF-1 mRNA confirmed vIRF-1 depletion in vIRF-1 (virf1) shRNA-expressing relative to non-silencing (ns) shRNA-expressing cells (left). (E) Equivalent analysis of ISG15 and ISG56 expression in iSLK cells infected with wild-type (wt) or vIRF-1-knockout (ttg) BAC16 virus and lytically reactivated with Dox/NaB treatment for 3 days.

Article Snippet: Primary antibodies used for immunoblotting were: S (Abcam, catalog number ab184223); CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); STAT1, pSTAT1, IRF9, His 6 , GST and p53 from Santa Cruz Biotechnologies (catalog numbers sc464, sc-365893, sc-8394, sc-803, sc-138, and sc-126, respectively); STAT2 and pSTAT2 from Cell Signaling Technology (catalog numbers 72604 and 4441).

Techniques: Transduction, Plasmid Preparation, Expressing, Control, shRNA, Derivative Assay, Biomarker Discovery, Infection, Knock-Out, Virus, Quantitative RT-PCR, Two Tailed Test

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) 293T cells transfected with empty vector (-) or vIRF-1 expression plasmid (virf1) were left untreated or treated with IFNβ or IFNγ (50 ng/ml of each) for 24 h. Cells were then harvested and lysates analyzed by immunoblotting for relative levels of pSTAT1 and pSTAT2, total STAT1 and STAT2, and IRF9. As applicable (detected), pSTAT1/STAT1 and pSTAT2/STAT2 ratios (“empty vector” values set at 1) are shown below the respective blots for IFNβ- and IFNγ-treated cells. (B) vIRF-1 suppression of IFNβ-induced signaling, as measured by ISRE-luciferase (luc) reporter assay, was detected in 293T cells cotransfected with the reporter and empty vector (-) or expression plasmid for vIRF-1 (virf1), left untreated or treated with IFNβ (50 ng/ml) for 24 h. Relative luciferase assay-derived luminescence values (RLU, means from duplicate transfectants) are shown along with standard deviations from the means. Student’s t-test P value (unpaired, two-tailed) for vIRF-1 suppression of IFNβ signaling (vIRF-1-vector relative to empty-vector RLU) is 0.0005. (C) STAT1, STAT2, and IRF9 knockout (KO) 293T cell lines were generated by Cas9/gRNA transduction, blasticidin selection, and clonal isolation of cells (see ). Target-gene ablation in each of the cell lines was tested functionally by ISRE-luciferase reporter assay following IFNβ stimulation (chart; N = 2). Mock, untreated cultures; virf1, vIRF-1 vector-transfected cells; -, empty vector-transfected cells. (D) vIRF-1 suppression of IFNβ-activated STAT1 and STAT2 (pSTAT1, pSTAT2) as a function of STAT1, STAT2, and IRF9 knockout. Cultures were transfected with empty vector (-) or vIRF-1 expression plasmid (virf1) and, after 6 h, treated with IFNβ (50 μg/ml) for 24 h. Cell lysates were analyzed by immunoblotting; numbers below the pSTAT1 and pSTAT2 blots show quantified pSTAT1 and pSTAT2 levels normalized to total STAT1 and STAT2, respectively, in the absence (value set at 1) and presence of vIRF-1. Arrows, STAT1/pSTAT1; asterisks, non-specific bands; dotted lines, lane deletions from single blot; solid line, different membrane.

Article Snippet: Primary antibodies used for immunoblotting were: S (Abcam, catalog number ab184223); CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); STAT1, pSTAT1, IRF9, His 6 , GST and p53 from Santa Cruz Biotechnologies (catalog numbers sc464, sc-365893, sc-8394, sc-803, sc-138, and sc-126, respectively); STAT2 and pSTAT2 from Cell Signaling Technology (catalog numbers 72604 and 4441).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Luciferase, Reporter Assay, Derivative Assay, Two Tailed Test, Knock-Out, Generated, Transduction, Selection, Isolation, Membrane

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) 293T cells transfected with empty vector (control) or vIRF-1 expression plasmid were left untreated (0 h) or treated with IFNβ (50 ng/ml) for different times (0.25 to 8 h). Cells were harvested, lysed, and analyzed by immunoblotting for detection of phosphorylated and total STAT1 and STAT2, IRF9, and also vIRF-1, to check expression in the transfectants. Relative levels of pSTAT1 and pSTAT2, normalized to STAT1 and STAT2, respectively, and with empty vector (vec) values at 0.25 h set at 1, are shown in the adjacent charts. (B) An analogous experiment was performed in STAT2-knockout (KO) 293T cells. The immunoblot-derived pSTAT1 levels, normalized to STAT1 and expressed relative to vector/0.25 h pSTAT1 (set at 1), are plotted in the chart (right). For both panels, dotted lines indicate lane deletion or rearrangement of blot sections for consistency of presentation.

Article Snippet: Primary antibodies used for immunoblotting were: S (Abcam, catalog number ab184223); CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); STAT1, pSTAT1, IRF9, His 6 , GST and p53 from Santa Cruz Biotechnologies (catalog numbers sc464, sc-365893, sc-8394, sc-803, sc-138, and sc-126, respectively); STAT2 and pSTAT2 from Cell Signaling Technology (catalog numbers 72604 and 4441).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Knock-Out, Derivative Assay

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: IFNβ binds to IFNAR1 and IFNAR2 to effect receptor activation through auto/cross-phosphorylation of receptor associated JAK kinases (JAK1 and TYK2) and receptor tyrosine residues. STAT1 and STAT2 are recruited and activated through JAK-mediated tyrosine phosphorylation, and then can heterodimerize and associate with IRF9 to form nuclear-localizing and transcriptionally active ISGF3 complexes. Data presented here show that vIRF-1 binds directly to STAT1, STAT2 and IRF9 and can also associate, directly or indirectly, with TYK2 and JAK1. vIRF-1 inhibits TYK2, but not JAK1, autophosphorylation (activation), likely contributing to pSTAT2 suppression; vIRF-1 also inhibits TYK2-STAT2 and TYK2-IFNAR1 association (not illustrated). However, suppression of pSTAT1 is mediated largely after IFNβ-induced STAT1 phosphorylation, involving increased rates of pSTAT1 decay, likely via phosphatase (P’ase)-mediated dephosphorylation (rather than pSTAT1 degradation). Suppression of pSTAT1 by vIRF-1 is dependent on STAT2, being negated by STAT2 knockout in 293T cells; this along with decreased IFNβ-stimulated pSTAT1 association with STAT2 and IRF9 in the presence of vIRF-1 indicates that vIRF-1 may suppress pSTAT1 via release of the transcription factor from ISGF3 or STAT1-STAT2 heterodimers, thereby promoting dephosphorylation of pSTAT1. Detection of intracellular complexes containing vIRF-1, pSTAT2 and IRF9 and dissociation of IFNβ-induced STAT1-STAT2 interaction by vIRF-1 in vitro are consistent with this model. Data from in vitro competition and STAT1/IRF9 and IRF9/STAT2 serial precipitation experiments provide evidence that vIRF-1 may also destabilizes IRF9-STAT2 association and dissociate ISGF3 interactions. It is possible that vIRF-1 also inhibits ISGF3 complex formation (not illustrated), via (detected) interactions with pre- and/or post-activated STAT1, STAT2 and IRF9, but the presented data do not specifically address this. In the diagram, blue lines indicate activities and consequences of vIRF-1.

Article Snippet: Primary antibodies used for immunoblotting were: S (Abcam, catalog number ab184223); CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); STAT1, pSTAT1, IRF9, His 6 , GST and p53 from Santa Cruz Biotechnologies (catalog numbers sc464, sc-365893, sc-8394, sc-803, sc-138, and sc-126, respectively); STAT2 and pSTAT2 from Cell Signaling Technology (catalog numbers 72604 and 4441).

Techniques: Activation Assay, Phospho-proteomics, De-Phosphorylation Assay, Knock-Out, In Vitro

Journal: Redox Biology

Article Title: ROS via BTK-p300-STAT1-PPARγ signaling activation mediates cholesterol crystals-induced CD36 expression and foam cell formation

doi: 10.1016/j.redox.2016.12.005

Figure Lengend Snippet: CC induces p300 activation in BTK-dependent manner. A. Equal amounts of protein from control and the indicated time periods of CC (40 μg/ml)-treated cells were immunoprecipitated with anti-p300 antibodies or IgG and the immunocomplexes were analyzed by Western blotting with anti-PY20 or anti-phosphoserine/threonine antibodies followed by normalization for p300. B. All the conditions were same as in panel A except that the cell extracts were analyzed by Western blotting for pBTK, pPyk2, pSrc and pSyk levels using their phospho-specific antibodies and normalized for their total levels. C. Cells were transfected with the indicated ASO, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in , panel C. The blot was reprobed for BTK, Pyk2 and β-tubulin levels to show the effects of the ASOs on their target and off target molecules levels. D. Cells were transfected with control or BTK ASO, quiesced, treated with and without CC for 1 h and cell extracts were prepared. Equal amount of protein from control and each treatment were immunoprecipitated with anti-p300 or anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for anti-PY20, anti-STAT1, anti-Ac-Lys or anti-PPARγ antibodies and normalized for p300 or STAT1. An equal amount of protein from control and each treatment was also assayed for p300 acetyltransferase activity. *p<0.01 vs control ASO; **p<0.01 vs control ASO+CC.

Article Snippet: Anti-pBTK (5082), anti-pPyk2 (3291), anti-pSrc (2101), anti-pSTAT1 (9177), anti-pSTAT2 (4441), anti-pSTAT3 (9133), anti-pSTAT4 (5267), anti-pSTAT5 (9351), anti-pSTAT6 (9364), and anti-pSyk (2715) antibodies were bought from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Control, Immunoprecipitation, Western Blot, Transfection, Expressing, Activity Assay