All too human?

Review of three books: 'Music and humanism: an essay in the aesthetics of music' by R A Sharpe; 'The spheres of music: a gathering of essays' by Leonard B Meyer; Critical entertainments: music old and new' by Charles Rosen, which appeared in Musical Times Autumn 2001

sj-pdf-1-vdi-10.1177_10406387221146247 – Supplemental material for Immunohistochemical analysis of expression of VEGFR2, KIT, PDGFR-β, and CDK4 in canine urothelial carcinoma

Supplemental material, sj-pdf-1-vdi-10.1177_10406387221146247 for Immunohistochemical analysis of expression of VEGFR2, KIT, PDGFR-β, and CDK4 in canine urothelial carcinoma by Laura C. Setyo, Shannon L. Donahoe, Patrick L. Shearer, Penghao Wang and Mark B. Krockenberger in Journal of Veterinary Diagnostic Investigation</p

MOESM1 of Cellular and molecular characterization of a stem rust resistance locus on wheat chromosome 7AL

Additional file 1. Histogram of read count. Bars represent the number of Unigene for a particular number of reads, and blue circles represent the additive percent of Unigene. Blue line represents the additive percent of reads

ERRα negatively regulates type I interferon induction by inhibiting TBK1-IRF3 interaction

<div><p>Estrogen-related receptor α (ERRα) is a member of the <a href="http://en.wikipedia.org/wiki/Nuclear_receptor" target="_blank">nuclear receptor</a> superfamily controlling energy homeostasis; however, its precise role in regulating antiviral innate immunity remains to be clarified. Here, we showed that ERRα deficiency conferred resistance to viral infection both <i>in vivo</i> and <i>in vitro</i>. Mechanistically, ERRα inhibited the production of type-I interferon (IFN-I) and the expression of multiple interferon-stimulated genes (ISGs). Furthermore, we found that viral infection induced TBK1-dependent ERRα stabilization, which in turn associated with TBK1 and IRF3 to impede the formation of TBK1-IRF3, IRF3 phosphorylation, IRF3 dimerization, and the DNA binding affinity of IRF3. The effect of ERRα on IFN-I production was independent of its transcriptional activity and PCG-1α. Notably, ERRα chemical inhibitor XCT790 has broad antiviral potency. This work not only identifies ERRα as a critical negative regulator of antiviral signaling, but also provides a potential target for future antiviral therapy.</p></div

ERRα chemical inhibitor XCT790 has broad antiviral potency.

<p>(A) qRT-PCR analysis of <i>IFNB</i>, <i>IFIT1</i> and <i>IP-10</i> mRNA expression in 293T cells infected with VSV (MOI = 1.0) in the presence or absence of 2.5 μM XCT790 for the indicated times. The data were normalized to the expression of the β-Actin reference gene. (B) Plaque assay of VSV loads in supernatants of 293T cells infected with VSV (MOI = 1.0) in the presence or absence of 2.5 μM XCT790 for the indicated times. (C) Immunoblotting analysis of VSV-G protein expression in supernatants of 293T cells infected with VSV (MOI = 1.0) in the presence of the indicated dose of XCT790 for the indicated times. (D) Representative images in 293T cells infected with NDV-GFP in the presence or absence of XCT790 for the indicated times. (E) Plaque assay of HSV-1 titers in the supernatants of 293T cells in the presence or absence of 5 μM XCT790 for the indicated times. (F) ELISA assay of HBsAg (left panel) or HBeAg (right panel) in the supernatants of HepG2.2.15 cells treated with DMSO or XCT790 for the indicated times. (G) Viral titer analysis of VSV production in lung, liver or spleen isolated from VSV infected mice treated with DMSO or XCT790 (n = 6 per group). Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.</p

TBK1 is indispensable for viral-induced ERRα stabilization.

<p>(A) Immunoblotting analysis of ERRα protein expression in siCtrl or siPGC-1α cells infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as the equal loading control. (B) Immunoblotting analysis of ERRα protein expression in 293T cells transfected with Flag-ERRα and HA-TBK1 or HA-IKKε. α-Tubulin was used as the equal loading control. (C) Immunoblotting analysis of ERRα protein expression in 293T cells infected with VSV (MOI = 1.0) in the presence or absence of 1 μM BX795 for the indicated times. α-Tubulin was used as the equal loading control. (D) Immunoblotting analysis of ERRα protein expression in WT and TBK1^CRISPR-/- cells infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as the equal loading control. (E) Immunoblotting analysis of ERRα protein expression in 293T cells infected with VSV (MOI = 1.0) and treated with CHX in the presence or absence of 1 μM BX795 for the indicated times. (F) qRT-PCR analysis of ERRα mRNA expression in 293T transfected with Flag-TBK1. The data were normalized to the expression of the β-actin reference gene. (G) Immunoblotting analysis of ERRα protein expression in 293T cells transfected with the indicated plasmids. α-Tubulin was used as the equal loading control. (H) Immunoblotting analysis of ERRα protein expression in WT and MAVS-KO MEFs infected with VSV (MOI = 1.0) for the indicated times. (I) ERRα responsive promoter luciferase activity assay in 293T cells transfected with HA-TBK1 and Flag- ERRα. (J) Immunoprecipitation analysis of K48 linked-ubiquitination of ERRα in 293T cells transfected with Flag- ERRα, HA-TBK1 or HA-TBK1 KD together with Myc-ubiquitin in the presence of MG132. Loading controls were shown in the lower panel of some Figures. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.</p

The transcriptional activity of ERRα is dispensable for its role in antiviral signaling.

<p>(A) ERRE promoter luciferase activity assay in 293T cells transfected with Flag-ERRα or Flag-ERRα CA mutant. (B-C) IFN-β (B) or ISRE promoter luciferase activity(C) assay in 293T cells transfected with Flag-ERRα or Flag-ERRα CA mutant and infected with VSV (MOI = 1.0) for 12 h. (D) IFN-β promoter luciferase activity assay in 293T cells transfected with HA-TBK1, Flag-ERRα or its mutants. (E) Plaque assay of VSV loads in supernatants from ERRα^CRISPR-/-cells transfected with Flag-ERRα or Flag-ERRα CA mutant followed by VSV (MOI = 1.0) infection for the indicated times. (F) Immunoprecipitation analysis in 293T cells transfected with HA-TBK1 and Flag-ERRα or its mutants. (G) IFN-β promoter luciferase activity assay in WT and A20^CRISPR-/- cells transfected with Flag-ERRα. (H) Immunoblotting assay in WT and A20^CRISPR-/- cells transfected with Flag-ERRα. Loading controls were shown in the lower panel of some Figures. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.</p

ERRα deficiency confers resistance to viral infection both <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Survival of 6-week-old WT and ERRα-KO mice given tail vein injections of VSV (1 x 10⁸ pfu/g) (n = 10 per group). (B) Plaque assay of VSV loads in sera, liver or lung isolated from WT and ERRα-KO mice given tail vein injections of VSV for 24 h (n = 6 per group). (C) Survival of 6-week-old WT and ERRα-KO mice given tail vein injections of HSV-1 (1 x 10⁷ pfu/g) (n = 10 per group). (D) Plaque assay of VSV loads in supernatants of WT and ERRα-KO BMDMs infected with VSV (MOI = 1.0) for the indicated times. (E) Immunoblotting analysis of ERRα and VSV-G protein levels in lysates of WT and ERRα-KO BMDMs infected with VSV (MOI = 1.0) for the indicated times. (F) Plaque assay of VSV loads in supernatants from control (shCtrl) or ERRα knockdown (shERRα-2) 293T cells followed by VSV (MOI = 1.0) infection for the indicated times. (G) Plaque assay of VSV loads in supernatants from control (siCtrl) or ERRα knockdown (siERRα) A549 cells followed by VSV (MOI = 1.0) infection for the indicated times. (H) Microscopy imaging of shCtrl or shERRα-2 293T cells infected with NDV-GFP (MOI = 1.0) for the indicated times. (I) Plaque assay of HSV-1 loads in supernatants of shCtrl or shERRα-2 293T cells infected with VSV (MOI = 1.0) for the indicated times. Loading controls were shown in the lower panel of some Figures. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.</p

ERRα is stabilized by viral infection.

<p>(A-E) Immunoblotting analysis of ERRα protein expression in 293T (A), BMDMs (B), THP-1(C), MEFs (D), and HeLa (E) infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as the equal loading control. (F-G) Immunoblotting analysis of ERRα protein expression in THP-1 cells infected with HSV (F) or incubated with 100 ng/ml LPS (G) for the indicated times. α-Tubulin was used as the equal loading control. (H) Immunoblotting analysis of ERRα protein expression from tissues of VSV-infected C57BL/6 mice collected at the indicated time points (n = 3 per group). α-Tubulin was used as the equal loading control. (I) Immunoblotting analysis of fractionated 293T cells infected with VSV for the indicated times. Cyt, cytosolic; Nuc, nuclear. The purity of the fractions was assessed by blotting for Lamin A (nuclear protein) and α-Tubulin (cytosolic protein). (J) qRT-PCR analysis of ERRα mRNA expression in 293T cells infected with VSV (MOI = 1.0) for the indicated times. The data were normalized to the expression of the β-actin reference gene. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.</p

ERRα negatively regulates IFN-I production both <i>in vitro</i> and <i>in vivo</i>.

<p>(A) ELISA analysis of IFN-β secretion in WT and ERRα-KO BMDMs transfected with Lipofectamine, 5’-ppp dsRNA (0.1 g/ml), poly (I:C) (0.1 g /ml), poly(dA:dT) (0.1 g /ml) and c-di-GMP (10 g /ml) or incubated with the Medium control (Med), HMW poly(I:C) (0.5 g/ml), LPS (0.1 g /ml) or flagellin (1 g /ml) for 12 h. (B) qRT-PCR analysis of IFN-β mRNA expression in WT and ERRα-KO BMDMs treated as shown in Fig 3A. (C-D) ELISA analysis of IFN-β (C) and qRT-PCR analysis of IFN-β mRNA (D) expression in WT and ERRα-KO BMDMs infected with VSV for the indicated hours. (E) IFN-β promoter luciferase activity assays in shCtrl or shERRα-2 293T cell lines infected with VSV (MOI = 1.0) for 12 h. (F) qRT-PCR analysis of IFN-β mRNA expression in lung, liver or spleen isolated from WT and ERRα-KO mice given tail vein injections of VSV for 24 h (n = 6 per group). (G) ELISA analysis of IFN-β protein in sera from WT and ERRα-KO mice given tail vein injections of VSV for 24 h (n = 6 per group). (H) Pathology of WT and ERRα-KO mice in response to VSV. Scale bar, 100 mm. HE staining of lung sections. Loading controls were shown in the lower panel of some Figures. Cell-based studies were performed independently at least three times with comparable results. The data are presented as the means ± SEM.</p