Given the apparent role of STAT3 in the biology of OSA, clinically relevant the

Pull down experiments within cells transfected with biotinylated STAT3 decoy ODN followed by western blotting showed that phos pho STAT3 interacted with STAT3 decoy ODN. In these experiments, the binding of phospho STAT3 to the STAT3 decoy ODN was blocked by the treatment of cells with Stattic, known to prevent STAT3 dimer formation, indicating that the STAT3 decoy ODN binds supplier AP24534 activated STAT3 dimers. Total STAT3 was almost exclusively nuclear in untreated cells, but after treat ment with STAT3 decoy ODN it became cytoplasmic. This was not observed when using mutated STAT3 decoy ODN but was observed when treating cells with Stattic. Subcellular locali zation of STAT3 studied by cell fractionnation and wes tern blotting showed that STAT3 decoy ODN efficiently prevented STAT3 nuclear translocation, whereas neither a mutated STAT3 decoy ODN, nor a NF B decoy ODN did so.

A diagram of the data collected from several experi ments illustrates the effect of STAT3 decoy ODN on the nucleo cytoplasmic distribution オーダー AT7519 of STAT3, in STAT3 decoy ODN transfected cells nuclear STAT3 consisted of 20% of the total STAT3, con versely, in control, mutated decoy ODN, and NF B decoy ODN transfected cells, it consisted of 50% of the total STAT3, suggesting that STAT3 decoy ODN func tions by trapping STAT3 within the cytoplasm. Finally, the binding of STAT3 to biotinylated STAT3 decoy ODN within cells was blocked by addition of excess non biotinylated STAT3 decoy ODN. Cytoplasmic sequestration of STAT3 and phospho STAT3 by STAT3 decoy ODN correlates with STAT3 inhibition and cell death In STAT3 decoy ODN transfected SW 480 cells, cell death increased, as previously shown in these and other cells.

STAT3 decoy ODN also induced cell death ATP-competitive Akt 阻害剤 of MCF 7 cells, in which a low but detectable STAT3 activation has been previously observed. However, the STAT3 decoy ODN had no effect on the acute myeloid leukemia cell line KG1, in which STAT5, rather than STAT3, is activated. The mutated STAT3 decoy ODN had no effect in any of the three cell lines. The STAT3 target cyclin D1 was analyzed by qPCR in STAT3 decoy ODN transfected SW 480 cells, reduced cyclin D1 expression was observed, similar to that observed with Stattic. This suggests that it is the cytoplasmic trapping of STAT3 by the STAT3 decoy ODN which leads to cell death.

Since STAT3 decoy ODN can also bind STAT1 and prevent STAT1 dependent IFNg induced cell death, experiments were performed to determine the overall involvement of STAT1 in STAT3 decoy ODN induced cell death. To this end, STAT1 was silenced using shRNA in the SW 480 cells. As previously described in other cell systems, the expression of STAT1 was suppressed by speci fic shRNA, and not by empty vector, NF B and STAT3 expression was unchanged. In STAT1 silenced SW 480 cells, the basal level of dead cells was unchanged. However, STAT3 decoy ODN induced cell death was suppressed. There was no effect of the mutated STAT3 decoy ODN, but IFNg induced cell death was suppressed. Thus, STAT1 expression is important for cell death induction by STAT3 inhibitors, in agreement with observations pub lished elsewhere, and in line with the notion that STAT1 is a key component of the cellular mechanism leading to cell death.