pshuttle2-socs1 viral vector  (Addgene inc)

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