engleski

Smads-mediated activation of p65/NF-kappaB and Smad- 4 nuclear translocation in U4C fibrosarcoma cells

No data has been reported on Smads mediated activation events in fibrosarcoma cells. To analyse the activation status of U4C fibrosarcoma cells upon transfection with different Smads constructs we have followed immunoreactivity of Smad-4 molecule in the nucleus by confocal microscopy. Thus, wild type U4C cells, cells deficient in Jak-1 molecule and overexpressing it have been transfected with Smad-1, Smad-2 or Smad 3 in order to see which signaling pathway - TGF- ( Smad-2 and Smad-3 mediated) or BMPs ( Smad-1 mediated) eventually become active We have observed high constitutive immunoreactivity of Smad-4 molecule in the nucleus of Jak-1 transgenic cells. This immunoreactivity has been reduced upon transfection with Smad 1, Smad-2 or Smad- 3. Also, the same cells have been stained with DAPI to evaluate apoptosis. Since NF-kappa B transcription factor has a dual role - pro and anti-apoptotic, DAPI stained cells were analysed for immunoreactivity of p65/NF-B subunit in the nucleus, a marker of NF-B activation. p65 subunit has been chosen to follow the translocation in to nucleus of both p52/p65 and p50/p65 NF-kappaB complexes. Western blot analysis revealed that both, wild type and genetically modified U4C cells, express p65, though, in genetically modified cells at slightly higher level.

Smads; NF-kappa B; Jak-1

nije evidentirano