plasmid expressing suppressor of cytokine signaling 3 (socs3) pcmv socs-3  (Addgene inc)

Journal: Scientific Reports

Article Title: Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation

doi: 10.1038/srep00637

Figure Lengend Snippet: Top 30 genes with promoters differentially methylated in control and miR122-silenced Huh7 cells. Values indicate methylation levels. Values in ‘Differences' indicate quantitative differences in methylation levels of target gene CpG sites. Higher values indicate greater methylation levels. Genes in bold and highlighted in gray means greater methylation; others were less methylated in miR122-silenced Huh7 cells

Article Snippet: Plasmids expressing Halo-tagged SOCS3 were purchased from Promega.

Techniques: Methylation

Journal: Scientific Reports

Article Title: Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation

doi: 10.1038/srep00637

Figure Lengend Snippet: (a) The CpG island in the SOCS3 promoter is indicated by double underlines. The numbers are the positions relative to the transcription start site. Primers used for the bisulphate sequences in this study are indicated by arrows. (b) Methylation status in the CpG island of the SOCS3 promoter in control and miR122-silenced Huh7 cells, determined by the bisulphate sequences. Seven clones each were sequenced. Circles represent CpG sites. Black circles, methylated CpG sites.

Article Snippet: Plasmids expressing Halo-tagged SOCS3 were purchased from Promega.

Techniques: Methylation, Clone Assay

Journal: Scientific Reports

Article Title: Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation

doi: 10.1038/srep00637

Figure Lengend Snippet: (a) Protein levels of the indicated IFN signaling-related genes were determined using control and miR122-silenced Huh7 cells after IFN-α, β, γ, or λ stimulation at the indicated time points. A representative of three independent determinations is shown. n.s. indicates non-specific bands. The band intensities were quantitated and adjusted by the expression levels of β-actin. The calculated ratios are indicated below each panel after setting the value of control cells at 0 h as 1.0. (b) Dnmt1 was not involved in the SOCS3 promoter methylation induced by miR122 silencing. SOCS3 protein levels were determined using control, miR122-silenced, Dnmt1 knocked-down, and Dnmt1 knocked-down with miR122-silenced Huh7 cell lysates. A representative of three independent determinations is shown. (c) MiR122 expression levels after IFN-λ stimulation for 6 h in Huh7 cells were determined by quantitative RT-PCR. Results were calculated by normalizing to U6 amounts and the relative ratio was determined by setting the value of unstimulated cells as 1. Data represent the mean ± SD of three independent determinations. *, p < 0.05.

Article Snippet: Plasmids expressing Halo-tagged SOCS3 were purchased from Promega.

Techniques: Expressing, Methylation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation

doi: 10.1038/srep00637

Figure Lengend Snippet: (a) HA-tagged SOCS3 was overexpressed in miR122-silenced Huh7 cells. Representative blotting images are shown. (b) SOCS3 overexpression suppresses enhanced ISRE activities in miR122-silenced Huh7 cells by a reporter assay. *, p < 0.05. Data represent the means ± SD of three independent determinations. (c) SOCS3 overexpression restored STAT2 and STAT3 phosphorylation levels induced by IFN-α in miR122-sileced Huh7 cells. A representative of three independent determinations is shown. The band intensities were quantitated and adjusted by the expression levels of β-actin. The calculated ratios are indicated below each panel after setting the value of control cells at 0 h as 1.0.

Article Snippet: Plasmids expressing Halo-tagged SOCS3 were purchased from Promega.

Techniques: Over Expression, Reporter Assay, Expressing