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A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

Inès Souissi12, Imen Najjar67, Laurent Ah-Koon12, Pierre Olivier Schischmanoff123, Denis Lesage12, Stéphanie Le Coquil12, Claudine Roger4, Isabelle Dusanter-Fourt67, Nadine Varin-Blank12, An Cao8, Valeri Metelev5, Fanny Baran-Marszak124 and Remi Fagard123*

Author Affiliations

1 INSERM, Unité 978, Bobigny, France

2 Université Paris 13, UFR SMBH, Bobigny, France

3 AP-HP, Hôpital Avicenne, Service de biochimie, Bobigny, France

4 AP-HP, Hôpital Avicenne, Service d'hématologie biologique, Bobigny, France

5 Moscow State University, Moscow, Russia

6 Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France

7 INSERM, Unité 1016, Paris, France

8 Groupe de Vectorisation, UFR SMBH, Université Paris 13, Bobigny, France

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BMC Cell Biology 2011, 12:14  doi:10.1186/1471-2121-12-14

Published: 12 April 2011

Additional files

Additional file 1:

Subcellular localization of the STAT3-decoy ODN. Cells were grown in 8-well plates to a density of 2.104 cells/mL. When the cells reached 50-60% confluence, they were transfected with the FITC-labeled (green) STAT3-decoy ODN (2 μg) in 150 μL of culture medium (DMEM without Fetal Calf Serum) combined to the liposomes (2 μg of cationic lipid). After 6 h at 37°C in a humidified 5% CO2 incubator, the cells were placed in fresh FCS-containing medium. After 48 h the cells were fixed and stained with DAPI to visualize nuclei and examined by fluorescence microscopy (A: nuclei, B: merge, C: FITC-labeled decoy).

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Additional file 2:

In-cell STAT3-decoy ODN pull-down assays. Cells were transfected with the STAT3-decoy ODN, as described under oligonucleotide transfection (see methods), and then processed by cell lysis and recovery on avidin-Sepharose beads. After extensive washing with binding buffer, complexes were separated on SDS-polyacrylamide (8%) gel, subjected to immunoblotting using an anti-phospho-STAT3 antibody (Cell Signaling); input was determined by analyzing an aliquot of the initial lysate with STAT3 antibody (Cell Signaling). Results were analyzed by chemiluminescence (LumiGLO, Cell Signaling) and autoradiography (X-Omat R, Kodak). In A, cells were either not treated (1) or treated with decoy STAT3-ODN (2). In B, cells were either not treated or treated with IL-6 (50 ng/ml).

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Additional file 3:

Effect of leptomycin B and of vanadate on the level of phospho-STAT3. Cells were either not treated (1, 2), treated with leptomycin B (LMB) (5 ng/ml) (3, 4), (10 ng/ml) (5, 6), (15 ng/ml) (7, 8) or vanadate (200 μM) (9, 10) (500 μM) (11, 12), for 4 h. Cytoplasmic (C) and nuclear extracts (N) (see methods) were analyzed on acrylamide gels and the membranes probed with anti-phospho-STAT3 and anti-Oct-1 antibodies.

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Additional file 4:

Effect of the STAT3-decoy ODN and of IL-6 on the nuclear localization of the p50 subunit of NF-κB. Cells were either not treated (1, 2), treated with STAT3-decoy ODN (2 μg/ml) (3, 4), IL-6 (50 ng/ml) (5, 6) or both (7, 8) for 6 h. Cytoplasmic (C) and nuclear extracts (N) (see methods) were analyzed on acrylamide gels and the membranes probed with anti-p50-NF-κB and anti-Oct-1 antibodies.

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