pcdna3 1 rat c ebp alpha  (Addgene inc)

Journal: Connective tissue research

Article Title: Histone Deacetylase Inhibitor Vorinostat (SAHA, MK0683) Perturb miR-9-MCPIP1 Axis To Block IL-1β-induced IL-6 Expression In Human OA Chondrocytes

doi: 10.1080/03008207.2016.1211113

Figure Lengend Snippet: (a) OA chondrocytes were stimulated with IL-1β (2ng/ml) in the presence or absence of SAHA (1μM) for 16 hrs. Chondrocytes were harvested and nuclei were isolated and subjected to nuclear run on assay as described above. Total RNA was isolated and MCPIP1 expression was quantitated using TaqMan assay (n=3). (b) OA chondrocytes were transfected with full length or deletion mutants of the MCPIP1 promoter reporter constructs and were stimulated with IL-1β alone or IL-1β + SAHA. Luciferase activity was measured after 24 hrs of stimulation. Renilla luciferase was used to normalize the transfection efficiency. (c) Diagramatic representation of the the156bp MCPIP1 promoter fragment with predicted transcription factors binding sites. (d) Transcription factor CEBPα was upregulated upon IL-1β and SAHA treatment determined by TaqMan assay and Western blot analysis. (*P <0.05; **P <0.005; paired student t-test).

Article Snippet: Luciferase Activity Assay For promoter analysis OA chondrocytes were transfected with the PCR generated MCPIP1 promoter fragments cloned into a luciferase reporter vector or with CEBPα expression plasmid (Addgene plasmid # 12550).

Techniques: Isolation, Nuclear Run-on Assay, Expressing, TaqMan Assay, Transfection, Construct, Luciferase, Activity Assay, Binding Assay, Western Blot

Journal: Connective tissue research

Article Title: Histone Deacetylase Inhibitor Vorinostat (SAHA, MK0683) Perturb miR-9-MCPIP1 Axis To Block IL-1β-induced IL-6 Expression In Human OA Chondrocytes

doi: 10.1080/03008207.2016.1211113

Figure Lengend Snippet: (a) OA chondrocytes were co-transfected with 156bp MCPIP1 wild type or mutant luciferase construct and CEBPα expression plasmid. Luciferase activity was measured and normalized against Renilla luciferase. (b) Endogenous MCPIP1 mRNA was measured by qPCR upon transfection of CEBPα in OA chondrocytes. (c) OA chondrocytes were transfected with non-targeted siRNA or siRNA against CEBPα. Twenty hours later chondrocytes were induced with 2 ng/ml IL-1β for 2 hrs and then treated with SAHA for an additional 14 hrs in serum free CellGro Active chondrocyte culture medium. Expression of CEBPα was determined using TaqMan Assay. (d) MCPIP1 expression was quantified by TaqMan Assay in OA chondrocytes transfected with non-targeted and CEBPα targeting siRNAs. (e) OA chondrocytes treated with IL-1β alone or with SAHA were subjected to ChIP analysis using anti-CEBPα antibody (# sc-61, Santa Cruz Biotechnology). Immunoprecipitated DNA was quantitated by qPCR and the values plotted against immunoprecipitated DNA from un-induced OA chondrocytes. (f) Expression of CEBPα mRNA was measured in smooth and damaged cartilage by TaqMan assay. (*P <0.05; **P <0.005; paired student t-test).

Article Snippet: Luciferase Activity Assay For promoter analysis OA chondrocytes were transfected with the PCR generated MCPIP1 promoter fragments cloned into a luciferase reporter vector or with CEBPα expression plasmid (Addgene plasmid # 12550).

Techniques: Transfection, Mutagenesis, Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, TaqMan Assay, Immunoprecipitation