Journal: European journal of immunology

Article Title: Functional involvement of Daxx in gp130-mediated cell growth and survival in BaF3 cells.

doi: 10.1002/eji.201040688

Figure Lengend Snippet: Figure 1. Involvement of Daxx protein in the regulation of gp130/ STAT3-dependent cell proliferation. (A) BaF-G133 cells were transfected with Daxx-specific and control siRNA and Daxx knockdown was validated by western blot analysis of Daxx, STAT3 and b-actin. Knockdown of Daxx was comfirmed until 3 days after siRNA transfection. Data are representative of three independent experi- ments. (B) G-CSF-induced cell proliferation of control or Daxx siRNA transfected BaF-G133 cells was determined by the WST8 assay. Data show mean7SD of triplicate samples and representative of three independent experiments. po0.05 and po0.01 (Student’s t-test). (C) Lysates from stable transfectants of BaF-G133 cells over-expressing Daxx were subjected to western blot analysis of Daxx and b-actin. Data are representative of three independent experiments. (D) The WST8 assay was performed on BaF-G133 cells and Daxx transfectants. Data show mean7SD of triplicate samples and representative of three independent experiments. po0.01 (Student’s t-test).

Article Snippet: The DNA-binding activity of STAT3 in cell extracts was measured using an immobilized STAT3 consensus oligonucleotide–Sepharose conjugate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as described previously [49].

Techniques: Transfection, Control, Knockdown, Western Blot, Expressing

Journal: European journal of immunology

Article Title: Functional involvement of Daxx in gp130-mediated cell growth and survival in BaF3 cells.

doi: 10.1002/eji.201040688

Figure Lengend Snippet: Figure 2. Daxx regulates cell cycle progression and cell death during gp130/STAT3-dependent cell growth. (A) BaF-G133 cells were treated with control siRNA or Daxx siRNA. Cells were IL-3-starved for 12 h and then treated with 30 ng/mL G-CSF for the indicated times. Cells were then fixed and subjected to cell cycle analysis using FACS. The percentages of cells in S plus G2/M phases of the cell cycle are indicated (mean7SD, n 5 3). po0.05 (Student’s t-test). (B) Daxx knockdown and control cells were cultured with the indicated concentrations of G-CSF for 48 h and the effect of Daxx knockdown on the G-CSF-induced reduction in cell death of BaF-G133 cells was evaluated by LDH activity in cell culture supernatants. Results are presented as the percentage of the maximum LDH release observed in untreated control cells (mean7SD, n 5 3). po0.01 (Student’s t-test). (C) The LDH assay was performed on BaF-G133 cells and Daxx transfectants, cultured with 30 ng/mL of G-CSF for 48 h. Results are presented as OD at 490 nm (Mean7SD, n 5 3). po0.01 (Student’s t-test). (D) BaF-G133 and BaF-G133/Daxx]3 cells were treated with 30 ng/mL G-CSF for the indicated times and the percentages of Annexin-V-positive cells were determined by FACS analysis (Mean7SD, n 5 3). po0.05 (Student’s t-test).

Article Snippet: The DNA-binding activity of STAT3 in cell extracts was measured using an immobilized STAT3 consensus oligonucleotide–Sepharose conjugate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as described previously [49].

Techniques: Control, Cell Cycle Assay, Knockdown, Cell Culture, Activity Assay, Lactate Dehydrogenase Assay

Journal: European journal of immunology

Article Title: Functional involvement of Daxx in gp130-mediated cell growth and survival in BaF3 cells.

doi: 10.1002/eji.201040688

Figure Lengend Snippet: Figure 3. Daxx interacts with STAT3 and inhibits its activity. (A) BaF-G133 cells (5 107 cells) were stimulated with G-CSF (30 ng/ml) for 30 min. The cells were lysed, immunoprecipitated with control IgG or anti-Daxx antibody and subjected to western blot analysis of STAT3 and Daxx. An aliquot of total cell extract (input) was also analyzed. (B) BaF-G133 cells (2 106 cells) were treated with control siRNA or Daxx siRNA, and cells were stimulated with G-CSF (30 ng/ml) for the indicated periods. Cell extracts were prepared and subjected to pull-down experiments using the immobilized STAT3 consensus oligonucleotide-sepharose conjugate to evaluate DNA binding activity of STAT3. The precipitates and an aliquot of total cell extract were subjected to western blot analysis of STAT3. (C) BaF-G133 cells (4 106 cells) were treated with control siRNA or Daxx siRNA and cells were stimulated with G-CSF (30 ng/ml) for the indicated periods. Samples for ChIP were prepared as described in the Materials and methods section. STAT3-DNA binding complexes were immunoprecipitated with the anti-STAT3 antibody or with control IgG. The immunoprecipitated DNA was eluted and subjected to PCR. (D) BaF-G133 cells were treated with control or Daxx siRNA and cells were stimulated with G-CSF (30 ng/mL) for 30 min. Total RNA samples isolated from these cells were subjected to quantitative RT-PCR analysis using Socs3 or Actb primers. Data represent the levels of Socs3 mRNA normalized to that of an Actb internal control and are expressed relative to the value of control siRNA-treated samples without G-CSF-stimulation. Results are representative of three independent, duplicate experiments. (E) BaF-G133 cells were treated with control or Daxx siRNA and cells were stimulated with G-CSF (30 ng/ml) for the indicated periods. Total RNA samples isolated from these cells were subjected to semi quantitative RT-PCR analysis using JunB, Pim2, Daxx or Gapdh primers. Data in (A), (C) and (E) are representative of three independent experiments; (B) is representative of two independent experiments.

Article Snippet: The DNA-binding activity of STAT3 in cell extracts was measured using an immobilized STAT3 consensus oligonucleotide–Sepharose conjugate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as described previously [49].

Techniques: Activity Assay, Immunoprecipitation, Control, Western Blot, Binding Assay, Isolation, Quantitative RT-PCR