Journal: BMC Molecular Biology

Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells

doi: 10.1186/1471-2199-15-24

Figure Lengend Snippet: DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

Article Snippet: Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

Techniques: DNA Methylation Assay, Amplification, Generated, Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Western Blot, Autoradiography

Journal:

Article Title: Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor ?-induced cell death in fibroblasts

doi:

Figure Lengend Snippet: SSI-1 relates to p38 MAP kinase, and not to JAK2, phosphatidylinositol 3-kinase–Akt, or MEK-ERK on TNF-α signaling. Sensitizing effect of various inhibitors on TNF-α-induced cell death in L929/SSI-1 or L929/neo. (A) L929/SSI-1 or L929/neo was pretreated for 2 h with AG490, an inhibitor of JAK2, at the indicated doses. Cells were then treated with TNF-α (0 or 1 ng/ml) for 24 h, at which time cell viability was determined by WST-8 and 1-Methoxy PMS and is shown as percentage of living cells and as mean ± SE. (B) Effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase. (C) Effects of U0126, an inhibitor of MEK1 and MEK2. (D) This histogram shows the effects of SB203580, an inhibitor of p38 MAP kinase.

Article Snippet: The activated form of p38 MAP kinase was immunoprecipitated with phospho-specific p38 MAP kinase (Thr 180/Tyr 182) antibody (NEB, Beverly, MA).

Techniques:

Journal:

Article Title: Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor ?-induced cell death in fibroblasts

doi:

Figure Lengend Snippet: SSI-1 sustains p38 MAP kinase signaling on TNF-α signals. (A) The phosphorylation of the recombinant activating transcription factor 2 (ATF2) substrate of p38 MAP kinase by in vitro kinase assay. L929/neo, SSI-1, SSI-3, SOCS-5, or SSI-1 R/Q mutant was treated with TNF-α (10 ng/ml) for the indicated time periods. Cells were harvested, and activated p38 MAP kinase was immunoprecipitated with anti-phosphorylated p38 MAP kinase. The activity of p38 MAP kinase was measured by means of immune complex kinase assays with ATF2 as the substrate (Lower). Control Western blots for levels of p38 MAP kinase protein are also shown (Upper). (B) In vitro kinase assay of p38 MAP kinase. SSI-1 +/+ MEFs or SSI-1 −/− MEFs were treated with TNF-α (10 ng/ml) for the indicated time periods. (C) In vitro kinase assay of p38 MAP kinase. L929/SSI-1 or L929/neo cells were treated with IL-1β (10 ng/ml) for the indicated time periods.

Article Snippet: The activated form of p38 MAP kinase was immunoprecipitated with phospho-specific p38 MAP kinase (Thr 180/Tyr 182) antibody (NEB, Beverly, MA).

Techniques: Recombinant, In Vitro, Kinase Assay, Mutagenesis, Immunoprecipitation, Activity Assay, Immune Complex Kinase Assay, Western Blot