Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: Microarray analysis shows that DF-1 have an attenuated innate response compared with CEFs. ( A ) Results from the microarray comparison between untreated DF-1 and CEFs. Pie charts represent the number of up- and down- regulated transcripts associated with different biological processes, assessed by Gene Ontology (GO) search and summarized according to their functions (PANTHER classification system). Scatter plots and Venn diagrams, showing extent of differential gene expression (Log2) and numbers of genes differentially regulated, respectively, in comparisons between CEFs and DF-1, either ( B ) treated with recombinant chIFN-α (1000 units/ml, 6 h), or ( C ) infected with IBDV (multiplicity of infection, MOI 5; 16 h). Scatter plots were generated using the Genespring scatter plot tool. Scatter plots show DF-1 (Y-axis) versus CEFs (X-axis) cells. The scale on the X- and Y-axis indicate expression levels (log2) and change in gene expression represented as a gradient of blue and red color for low- and high-expression intensity respectively. ( D ) Cluster analysis and heat-map showing differential expression of the top 45 ISGs (|fold change| ≥3.0 and FDR ≤0.01) as identified in CEFs following chIFN-α stimulation. ISGs were ranked by hierarchical clustering. Columns represent five comparisons, left to right: IBDV-infected CEFs and DF-1, chIFN-α-stimulated CEFs and DF-1 (each compared to their respective mock-treated control) and mock-treated DF-1 compared to mock-treated CEFs. Fold change in gene expression is represented by a blue (down-regulated) to red (up-regulated) colour intensity gradient. ( E ) Microarray expression data (log2 normalised intensity values ± Standard Deviation) for SOCS1 in, IBDV-infected, chIFN-α –treated or mock-treated CEFs or DF-1.

Article Snippet: Cells were transfected with 1 μg of a SOCS1 expression vector using the jetPrime transfection reagent (Polyplus Transfection SA, Illkirch, France) according to manufacturers instructions for 48 hours at 37 °C and 5% CO 2 .

Techniques: Microarray, Comparison, Expressing, Recombinant, Infection, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: Relative suppression or induction of ISG transcription by overexpression or knockdown, respectively, of SOCS1. ( A – C ) DF-1 were transfected with control siRNA or siRNA specific for SOCS1 for 42 h and mock-treated or treated with chIFN-α (1000 units/ml) for 6 h. ( D – F ) DF-1 were transfected with either empty vector or an HA-tagged SOCS1 expression plasmid (SOCS1p) for 42 h and and mock-treated or treated with chIFN-α (1000 units/ml) for 6 h. Extracted total RNA was subjected to reverse transcription followed by quantitative PCR using specific primer sets for SOCS1 (A and D), Mx1 (B and E), IFIT5 (C and F) normalized against GAPDH (using the ΔΔCt method). Data in A-F are representative from three independent experiments. One-way ( A–F ) Anova with Bonferroni posthoc test were used to analyse the data. * P < 0.05, *** P < 0.001, **** P < 0.0001. ( G ) & ( H ) Immunoblots confirming expression of exogenous HA-tagged SOCS1 following transfection of DF-1 with either pcDNA4 (empty vector) or pcDNA4 expressing HA-tagged SOCS1 ( G ) and silencing of SOCS1 following transfection of DF-1 with the Flag-tagged SOCS1 construct and either siRNA for SOCS1 or a control siRNA ( H ). Panels (G) and (H) show cropped images of the immunoblots; full-length blots are presented in Supplementary Fig. .

Article Snippet: Cells were transfected with 1 μg of a SOCS1 expression vector using the jetPrime transfection reagent (Polyplus Transfection SA, Illkirch, France) according to manufacturers instructions for 48 hours at 37 °C and 5% CO 2 .

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Construct

Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: The SOCS box is not essential for ChSOCS1 inhibition of JAK/STAT signalling in DF-1. ( A ) Schematic representation of the architecture of SOCS1 protein, expression plasmids encoding wild-type (SOCS1 WT) and SOCS box deletion mutant (SOCS1 ΔBOX) and alignment of sequences (in boxes) of the KIR motif and the SOCS box domain of human, mouse and chicken SOCS1 proteins. ( B ) Luciferase reporter gene assay in DF-1 for chicken Mx1 and viperin promoters following transient transfection expression plasmids SOCS1 WT and SOCS1 ΔBOX, each with and without chIFN-α treatment. Two-way Anova with Bonferroni posthoc test were used to analyse the data. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( C ) Immunoblot confirming expression of Flag-tag in Flag-tagged expression plasmids encoding for wild-type (WT) SOCS1 and SOCS box deletion mutant (ΔBOX). No protein was observed when DF-1 were transfected with SOCS1 KIR deletion mutant (ΔKIR) plasmid. Panel (C) shows cropped images of the immunoblots; full-length blots are presented in Supplementary Fig. .

Article Snippet: Cells were transfected with 1 μg of a SOCS1 expression vector using the jetPrime transfection reagent (Polyplus Transfection SA, Illkirch, France) according to manufacturers instructions for 48 hours at 37 °C and 5% CO 2 .

Techniques: Inhibition, Expressing, Mutagenesis, Luciferase, Reporter Gene Assay, Transfection, Western Blot, FLAG-tag, Plasmid Preparation

Journal: Scientific Reports

Article Title: Constitutively elevated levels of SOCS1 suppress innate responses in DF-1 immortalised chicken fibroblast cells

doi: 10.1038/s41598-017-17730-2

Figure Lengend Snippet: Modulating levels of SOCS1 in CEFs and DF-1 regulates viral RNA expression and virus yield. ( A ) Titres of IBDV PBG98 recovered from CEFs and DF-1 after transient transfection with a SOCS1 expression plasmid or siSOCS1, respectively. Virus titres are the sum of cell-associated and extracellular virus, determined by plaque assay on CEFs (Mean ± SEM). ( B ) Fold change (percent) of IBDV VP4 RNA levels in CEFs and DF-1 after transient transfection with a SOCS1 expression plasmid or siSOCS1, respectively. Virus titres and viral RNA levels were compared with those from cells transfected with empty vectors or control siRNA, as appropriate. Data are representative from three independent experiments. An unpaired t-test with Welch’s correction (Two-tailed) was used to analyse the data. ** P < 0.01, **** P < 0.0001.

Article Snippet: Cells were transfected with 1 μg of a SOCS1 expression vector using the jetPrime transfection reagent (Polyplus Transfection SA, Illkirch, France) according to manufacturers instructions for 48 hours at 37 °C and 5% CO 2 .

Techniques: RNA Expression, Virus, Transfection, Expressing, Plasmid Preparation, Plaque Assay, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes

doi: 10.1038/emm.2016.47

Figure Lengend Snippet: Changes in MMP transcripts induced by SOCS1 overexpression and knockdown. ( a ) Compared with scramble shRNA-transfected cells (white bars), SW 1353 cells with SOCS1 knockdown (gray bars) showed a significant increase in MMP-3 and MMP-13 mRNA at 24 h. ( b ) Conversely, SOCS1-overexpressing SW1353 cells (black bars) produced significantly lower levels of MMP-3 and MMP-13 mRNA at 24 h after IL-1β stimuli. The levels of MMP-1 transcript were not affected by up- or downregulation of SOCS1 at 24 h. Data are expressed as the mean±s.e.m. (all n =3). * P <0.05.

Article Snippet: pBABE-C/EBPβ and the pShuttle2-SOCS1 viral vector were obtained from Addgene (Cambridge, MA, USA), and the pBABE-SOCS1 viral vector was generated as previously reported.

Techniques: Over Expression, shRNA, Transfection, Produced

Journal: Experimental & Molecular Medicine

Article Title: SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes

doi: 10.1038/emm.2016.47

Figure Lengend Snippet: The effect of SOCS1 on IL-1β-induced C/EBPβ expression. ( a ) IL-1β-induced C/EBPβ mRNA expression was significantly increased in SW1353 cells with knockdown of the SOCS1 gene (gray bars), and decreased in the SOCS1-overexpressing SW1353 cells after 8 h (black bars; n =3). ( b ) A representative immunoblot showing that IL-1β-induced expression of the C/EBPβ protein was lower in SOCS1-overexpressing SW1353 cells than in other cells (lane 1, empty vector; lane 2, SOCS1 vector; lane 3, scramble shRNA; lane 4, SOCS1 shRNA). ( c ) SOCS1 decreased the levels of IL-1β-induced C/EBPβ transcript and protein expression in human articular chondrocytes (HACs) electrotransfected with the SOCS1 vector ( n =5). ( d ) C/EBPβ knockdown or overexpression did not affect the levels of SOCS1 mRNA upon IL-1β stimulation ( n =3). Data are expressed as the mean±s.e.m. * P <0.05.

Article Snippet: pBABE-C/EBPβ and the pShuttle2-SOCS1 viral vector were obtained from Addgene (Cambridge, MA, USA), and the pBABE-SOCS1 viral vector was generated as previously reported.

Techniques: Expressing, Western Blot, Plasmid Preparation, shRNA, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes

doi: 10.1038/emm.2016.47

Figure Lengend Snippet: Post-translational ubiquitination of C/EBPβ by SOCS1. ( a ) SOCS1-knockdown or -overexpressing SW1353 cells were incubated with IL-1β (10 ng ml −1 ) for 24 h, lysed and subjected to immunoprecipitation (IP) using antibodies to C/EBPβ. The precipitates were analyzed by immunoblot with antibodies to ubiquitin. The lysates before IP were subjected to immunoblot analysis with antibodies to C/EBPβ. In C/EBPβ-siRNA-transfected SW1353 cells, the disappearance of ubiquitinated C/EBPβ was confirmed. A representative figure and densitometric analysis demonstrated that C/EBPβ ubiquitination was increased by SOCS1 overexpression and tended to be decreased by SOCS1 knockdown. ( b ) Treatment with MG132, a proteasome inhibitor, led to increased endogenous C/EBPβ protein expression after 24 h in SW1353 cells, regardless of SOCS1 expression status. ( c ) IL-1β stimulation of SOCS1-overexpressing SW1353 cells led to reduced C/EBPβ protein levels, compared with empty vector-transfected cells, even with the treatment of MG132. The data presented are representative of three independent experiments. * P <0.05 compared with empty vector-transfected SW1353 cells.

Article Snippet: pBABE-C/EBPβ and the pShuttle2-SOCS1 viral vector were obtained from Addgene (Cambridge, MA, USA), and the pBABE-SOCS1 viral vector was generated as previously reported.

Techniques: Incubation, Immunoprecipitation, Western Blot, Transfection, Over Expression, Expressing, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes

doi: 10.1038/emm.2016.47

Figure Lengend Snippet: The effect of SOCS1 on CREB phosphorylation. ( a ) IL-1β stimulation led to significantly increased amounts of phospho-CREB protein at 15–60 min post stimulation in SOCS1-knockdown SW1353 cells (gray bars) compared with control shRNA-transfected cells (white bars). ( b ) In contrast, phospho-CREB levels were significantly lower at 5–15 min in SOCS1-overexpressing SW1353 cells (black bars). The densitometry data were normalized to β-actin and expressed as the means±s.e.m. ( n =3). * P <0.05.

Article Snippet: pBABE-C/EBPβ and the pShuttle2-SOCS1 viral vector were obtained from Addgene (Cambridge, MA, USA), and the pBABE-SOCS1 viral vector was generated as previously reported.

Techniques: shRNA, Transfection

Journal: Experimental & Molecular Medicine

Article Title: SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes

doi: 10.1038/emm.2016.47

Figure Lengend Snippet: The involvement of a p38 MAPK on CREB phosphorylation and the interaction between HIF-2α and SOCS1. ( a ) Pretreatment with SB202190 suppressed IL-1β (10 ng ml −1 ) induced expression of phospho-CREB in a dose-dependent manner. In addition, in SOCS1-overexpressing SW1353 cells, IL-1β-induced CREB phosphorylation was further increased by adding anisomycin (ANS) as a p38 kinase inhibitor. ( b ) Under normoxic conditions, IL-1β induced C/EBPβ expression, but not HIF-2α expression. Hypoxia-induced HIF-2α expression was unchanged by SOCS1. The densitometry data were normalized to β-actin and expressed as the means±s.e.m. ( n =3).

Article Snippet: pBABE-C/EBPβ and the pShuttle2-SOCS1 viral vector were obtained from Addgene (Cambridge, MA, USA), and the pBABE-SOCS1 viral vector was generated as previously reported.

Techniques: Expressing

Journal: Experimental & Molecular Medicine

Article Title: SOCS1 suppresses IL-1β-induced C/EBPβ expression via transcriptional regulation in human chondrocytes

doi: 10.1038/emm.2016.47

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) assay of C/EBPβ binding to the MMP-13 promoter. ( a ) Binding of C/EBPβ to the MMP-13 promoter was reduced in SOCS1-overexpressing SW1353 cells under IL-1β stimulation (10 ng ml −1 , right panel), compared with empty vector-transfected cells. ( b ) Semiquantitative densitometric analysis of ChIP data showed that SOCS1 significantly decreased C/EBPβ binding to the MMP-13 promoter under IL-1β stimulation. The relative intensity of C/EBPβ binding to the MMP-13 promoter was normalized to PCR-amplified input DNA (10%) in each case. The error bars represent the s.e.m. from three independent measurements. * P <0.05.

Article Snippet: pBABE-C/EBPβ and the pShuttle2-SOCS1 viral vector were obtained from Addgene (Cambridge, MA, USA), and the pBABE-SOCS1 viral vector was generated as previously reported.

Techniques: Chromatin Immunoprecipitation, Binding Assay, Plasmid Preparation, Transfection, Amplification

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Primers used in cloning in this study

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Cloning, Sequencing

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Quantitative RT-PCR primers used in this study

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Quantitative RT-PCR, Sequencing

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: PRRSV infection induces SOCS1 expression in porcine AMs and Marc-145 cells. (A–C) Porcine AMs were infected or mock infected with HN07-1 at an MOI of 1. At the indicated time points, the cells were collected to assess SOCS1 mRNA levels (A) using qRT-PCR or SOCS1 protein levels (B) by Western blotting and ELISA (C). For ELISA, data were expressed as picograms per microgram of total protein. (D) Porcine AMs were infected or mock infected with HN07-1 at different MOIs. At 12 hpi, the cells were collected for analysis of SOCS1 mRNA levels using qRT-PCR. (E and F) Marc-145 cells were infected or mock infected with HN07-1 at an MOI of 1. At the indicated time points, the cells were collected, SOCS1 mRNA levels (E) were assessed using qRT-PCR, and SOCS1 protein levels (F) were assessed using Western blotting. (G and H) Porcine AMs were infected or mock infected with HNhx (G) or BJ-4 (H) at an MOI of 1. At the indicated times, the cells were collected, and SOCS1 mRNA levels were assessed using qRT-PCR. (I) Porcine AMs were infected or mock infected with HN07-1, heat-inactivated HN07-1, or UV-inactivated HN07-1. At the indicated time points, the cells were collected, and SOCS1 mRNA levels were assessed using qRT-PCR. All experiments were performed with at least three independent replicates. The results obtained were compared with 0 h (medium control) using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: SOCS1 enhances PRRSV replication. (A–D) Porcine AMs were transfected with 20 nM NC siRNA or siRNAs against SOCS1 (an equimolar mixture of two siRNAs) for 24 h before infecting them with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested, SOCS1 mRNA levels (A) were assessed using qRT-PCR, and SOCS1 protein levels (B) were assessed using ELISA. The cells were collected for PRRSV ORF7 (C) quantification using qRT-PCR or viral enumeration (D) using a TCID50 assay. (E and F) Marc-145 cells (E) and CRL2843-CD163 cells (F) were transfected with pcDNA3.1-Myc/his_A or pcDNA3.1-SOCS1-His and then infected 24 h later with HN07-1 (MOI = 0.1). At 24 hpi, the cells were collected, and SOCS1 protein levels were assessed using Western blotting with an anti-His Ab. Viruses (E) were enumerated using a TCID50 assay, and PRRSV ORF7 (F) was quantified using qRT-PCR. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significance was denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, TCID50 Assay, Infection, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Induction of SOCS1 expression by PRRSV is not completely dependent on type I IFNs. (A and B) Porcine AMs were treated or mock treated with B18R for 1 h and then treated with medium alone (control), HN07-1 (MOI = 1), or poly(I:C) at a final concentration of 10 μg/ml. Twenty-four hours later, porcine AMs were harvested and analyzed for SOCS1 mRNA (A) quantification using qRT-PCR and SOCS1 protein (B) quantification using ELISA. (C–E) Porcine AMs were transfected with 20 nM NC siRNA or siRNA against IFNAR1 for 24 h and then infected with PRRSV (MOI = 1). At 24 hpi, porcine AMs were harvested and analyzed for IFNAR1 (C) and SOCS1 mRNA (D) quantification using qRT-PCR or SOCS1 protein (E) quantification using ELISA. (F) Marc-145 cells were pretreated or mock treated with B18R for 1 h and then treated with medium alone (control), HN07-1 (MOI = 1), or poly(I:C) at a final concentration of 10 μg/ml. Twenty-four hours later, the cells were harvested and analyzed for SOCS1 mRNA quantification using qRT-PCR. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Expressing, Control, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Infection

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Cloning and sequence analysis of porcine SOCS1 promoter and mapping of potential transcriptional regulatory elements. (A) Schematic representation of the porcine SOCS1 promoter. SOCS1 promoter–truncated mutants were subcloned into pGL4.17-basic vector and the resulting constructs denoted as −2000/100-Luc, −1359/100-Luc, −381/100-Luc, and −58/100-Luc. The relative lengths and positions of the 5′ ends of these fragments were indicated. (B and C) Marc-145 cells (B) or CRL2843-CD163 cells (C) were transfected with a series of SOCS1 promoter–truncated mutants or pGL4.17-basic vector and a pRL-TK Renilla luciferase reporter plasmid. Twenty-four hours later, they were infected or mock infected with HN07-1 (MOI = 1). Another 24 h later, the cells were harvested to determine luciferase activity. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Cloning, Sequencing, Plasmid Preparation, Construct, Transfection, Luciferase, Infection, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: PRRSV N protein induces SOCS1 production. (A and D) Marc-145 cells (A) or CRL2843-CD163 (D) was cotransfected with a series of plasmids that encode PRRSV nsps and structural protein (nsp1α, nsp1β, nsp2, nsp4, nsp5, nsp11, and N) or pcDNA 3.1-Myc/his_A vector, −381/100-Luc promoter, and pRL-TK Renilla luciferase reporter plasmid. After 24 h, the cells were harvested for luciferase activity analysis. (B) Marc-145 cells were transfected with N protein expression vector at doses of 0.5, 1.0, and 2.0 μg per well (six-well plate) and were harvested at 48 h posttransfection. Western blotting using an anti-SOCS1 Ab was performed to assess SOCS1 protein levels. (C and E) Marc 145 cells (C) or CRL2843-CD163 (E) was cotransfected with PRRSV N protein deletion mutants or pcDNA3.1-Myc/His_A vector, −381/100-Luc promoter, and pRL-TK Renilla luciferase reporter plasmid. After 24 h, the cells were harvested for luciferase activity analysis. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: AP-1 binding element in the porcine SOCS1 promoter is indispensable for SOCS1 production. (A) Schematic diagram representing porcine SOCS1 promoter deletion mutants (−308ΔIRF3-Luc, −188ΔSp1-Luc, −160ΔSp1-Luc, −116ΔIRF3-Luc, −104ΔAP-1-Luc, and −381/100-Luc). (B and D) Marc-145 cells (B) or CRL2843-CD163 cells (D) were cotransfected with a series of SOCS1 promoter mutants and pRL-TK Renilla luciferase reporter plasmid for 24 h and then infected or mock infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for luciferase activity analysis. (C and E) Marc-145 cells (C) or CRL2843-CD163 cells (E) were cotransfected with a series of SOCS1 promoter mutants, N protein, and pRL-TK Renilla luciferase reporter plasmid. Twenty-four hours later, the cells were harvested for luciferase activity analysis. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Binding Assay, Luciferase, Plasmid Preparation, Infection, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: The AP-1 signaling pathway is involved in SOCS1 upregulation by PRRSV. (A) Porcine AMs were pretreated with DMSO or SR11302 for 1 h and then infected or mock infected with HN07-1 (MOI = 1) in the presence of inhibitor. The cells were harvested at 24 hpi for analysis of SOCS1 mRNA levels using qRT-PCR. (B–E) Porcine AMs were transfected with 20 nM NC siRNA or siRNAs against porcine c-Jun and c-Fos using RNAiMAX and then infected after 24 h with PRRSV HN07-1 (MOI = 1). After 24 h, the cells were harvested and analyzed to measure knockdown efficiency (B and C). Or the cells were harvested for analysis of SOCS1 mRNA levels (D) using qRT-PCR or lysed for SOCS1 protein (E) quantification using ELISA. (F) Marc-145 cells were pretreated with DMSO or SR11302 for 1 h and transfected with a vector that encodes the PRRSV N protein. After 24 h, the cells were harvested for SOCS1 protein level and p–c-Jun/c-Jun and p–c-Fos/c-Fos level quantification using Western blotting. (G) Porcine AMs were infected with HN07-1 (MOI = 1) and then harvested at the indicated time points for analysis of SOCS1, p–c-Jun/c-Jun, and p–c-Fos/c-Fos levels, using Western blotting. (H) Marc-145 cells were transfected with a vector that encodes the PRRSV N protein at doses of 0.5, 1.0, and 2.0 μg per well (six-well pates) and then harvested 48 h later for analysis of SOCS1 and p–c-Jun/c-Jun levels using Western blotting. Relative expression levels shown below the images were evaluated as fold changes after normalization to β-actin or GAPDH levels. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Infection, Quantitative RT-PCR, Transfection, Knockdown, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: The p38 and JNK MAPK signaling pathways are required for PRRSV upregulation of SOCS1 expression. (A and B) Porcine AMs were pretreated with DMSO, p38 inhibitor SB203580 (SB), ERK inhibitor PD98059 (PD), or JNK inhibitor SP600125 (SP) for 1 h and then infected or mock infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of SOCS1 mRNA levels (A) using qRT-PCR and SOCS1 protein levels (B) using ELISA. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by **p < 0.01.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Protein-Protein interactions, Expressing, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: The p38/AP-1 and JNK/AP-1 signaling pathways are involved in PRRSV-induced SOCS1 production. (A–C) Porcine AMs were pretreated with DMSO or p38 inhibitor SB203580 (SB) for 1 h and then infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of p–c-Jun/c-Jun (A), p–c-Fos/c-Fos (B), and SOCS1 levels (C) using Western blotting. (D–F) Porcine AMs were pretreated with DMSO or JNK inhibitor SP600125 (SP) for 1 h and then infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of p–c-Jun/c-Jun (D), p–c-Fos/c-Fos (E), and SOCS1 levels (F) using Western blotting. (G and H) Marc-145 cells were pretreated with DMSO, p38 inhibitor SB, or JNK inhibitor SP for 1 h, and they were then transfected with plasmid that encodes the PRRSV N protein at a dose of 2.0 μg per well (six-well plates) for 48 h. The cells were harvested for analysis of SOCS1 and p–c-Jun/c-Jun expression using Western blotting. Relative expression levels shown below the images were evaluated as fold changes after normalization to GAPDH levels.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Protein-Protein interactions, Infection, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: HAGE enhances SOCS1 RNA unwinding and protein expression. ( a ) Schematic representation of the mechanism of action of SOCS1. ( b ) Left panel: IF with antibodies to SOCS1 (green) and HAGE (red) on FM82 control and FM82 shRNA1 and 2 (HAGE knocked down). Right panel: IB with antibodies to HAGE, SOCS1 and actin (loading control) using whole lysates from FM82 and FM55 control and FM82 and FM55 shRNA1 and 2. Scale bar 50 μ m. ( c ) Representative image of immunohistochemistry with antibodies to SOCS1 (green) and HAGE (red) on normal skin and malignant melanoma sections. Scale bar 100 μ m. ( d ) IB with antibodies to HAGE, SOCS1, p-Jak1, p-Tyk2, PML and actin using whole cell extracts from FM82 and FM55 control and FM82 and FM55 shRNA1 and 2 transfected with siRNA control and siRNA for SOCS1 mRNA. ( e ) Transient silencing of SOCS1 in HAGE stable knockdown and control FM82 cells followed by rescue of HAGE expression using a HAGE cDNA expression vector. Actin was measured as a loading control. ( f ) Semi-quantitative real-time PCR of SOCS1 mRNAs isolated from FM82 control and FM82 shRNA1. ( g ) Unwinding of biotinylated SOCS1 N-terminal complimentary RNA sequence-SOCS1 RNA duplexes in the presence of increasing HAGE protein concentrations (lane 1: 0 μ g, lane 2: 0.6 μ g, lane 3: 1.2 μ g). Biotinylated SOCS1 N-terminal complimentary RNA sequence was used as a loading control in lane 4. ( h ) IP with antibody to SOCS1 of biotin alkyle metabolically labelled (30, 60 and 120 min) and unlabelled SOCS1 proteins followed by IB with streptavidin antibody from FM82 control whole cell extracts transfected with SOCS1cDNA and with or without transfected HAGE cDNA

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: Expressing, Immunohistochemistry, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Isolation, Sequencing, Metabolic Labelling

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: SOCS1 induces ubiquitination of p-Jak1 and p-Tyk2 in FM82 control malignant melanoma cell lines. ( a ) IP with antibody to SOCS1 or rabbit IgG followed by IB with antibodies to SOCS1, p-Jak1 and p-Tyk2 from FM82 control whole cell extracts. ( b and c ) IP with antibody to p-Jak1 or p-Tyk2 and corresponding IgG (rabbit or goat, respectively) followed by IB with antibodies to p-Jak1 or p-Tyk2 and ubiquitin from FM82 control and FM82 shRNA1 whole cell extracts. ( d ) IP with antibody to p-Jak1 followed by whole cell extracts IB with an antibody to ubiquitin from FM82 shRNA1 transfected with HAGE cDNA or SOCS1 cDNA. The protein expression of HAGE and SOCS1 was determined by IB with the corresponding antibodies

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: Transfection, Expressing

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: HAGE counteracts the anti-proliferative effect of IFN α in ABCB5+ MMICs. ( a , b and c ) IF with antibodies to HAGE (red), ABCB5 (green), SOCS1 (green) and PML (green) on FM82 control, FM82 shRNA1 and 2 melanoma spheres. ( d ) IF and IB with an antibody to PML (or IgG) on FM82 cell lines and corresponding melanoma spheres stably-expressing PML or the empty vector. ( e ) FM82 empty vector and stably expressing PML spheres formation in the presence or absence of cDNA transfected HAGE or SOCS1. ANOVA: *** P =<0.0001. Scale bar 100 μ m (for spheres) and 20 μ m (for cells). ( f ) IF and IB with an antibody to PML on FM82 shRNA/PMLshRNA CTL and FM82shRNA/PMLshRNA 1 and 2. ( g ) Spheres formation assay from FM82shRNA/PMLshRNA CTL and FM82shRNA/PML shRNA 1 and 2. ANOVA: *** P =<0.0001

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, shRNA, Tube Formation Assay

Journal: Cell Death & Disease

Article Title: The helicase HAGE prevents interferon- α -induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

doi: 10.1038/cddis.2014.29

Figure Lengend Snippet: HAGE counteracts the anti-proliferative effect of IFN α in vivo . ( a , b and c ) FM82 and FM55 controls and FM82 and FM55 shRNA 1 and 2 melanoma sphere formation in the presence or absence of IFN α . Student t -test: ** P =<0.01. ( d ) Summary of the in vivo experimental procedure. ( e ) Measurement of tumour growth in mice injected with either FM82 control cells or FM82 shRNA1 cells and treated or untreated with IFN α ANOVA: ** P =<0.0028; Mann–Whitney U -test: * P =<0.0108 (when comparing FM82 control + PBS and FM82 shRNA + PBS; Mann–Whitney U -test: ** P =<0.0079 (when comparing FM82 control + IFN α and FM82 shRNA + IFN α treated). ( f ) Immunohistochemistry/fluorescence staining with antibodies to HAGE (red), ABCB5 (green), SOCS1 (green) and on sections from FM82 control and FM82 shRNA1 NOD/SCID xenotransplanted tumours and treated or untreated with IFN α . Scale bar 100 μ m

Article Snippet: HAGE or SOCS1 ectopic expression were carried out using pCMV6-DLX5-HAGE (SC126051, OriGene, Rockville, MD, USA) and pCMV6-XL4-SOCS1 (SC111081, Origene).

Techniques: In Vivo, shRNA, Injection, MANN-WHITNEY, Immunohistochemistry, Fluorescence, Staining