Figure 4: Nuclear localization of SMAD3/4 in TGF-β stimulated CF.
From: Fibulin-6 regulates pro-fibrotic TGF-β responses in neonatal mouse ventricular cardiac fibroblasts
nCF transfected with scr siRNA or Fibulin-6 siRNA, with and without TGF-β stimulation are fixed and stained for SMAD3 expression or are processed for nuclear lysate preparation. (a) Cells are stained with SMAD3 (red) and DAPI (blue) (Scale bar = 100 μm). In scr siRNA transfected CF translocation of SMAD3 into the nucleus can be observed upon TGF-β stimulation. Arrows indicate SMAD3 positive nuclei. In fibulin-6 KD CF SMAD3 staining is almost absent in nuclei. High magnification inserts show nuclear SMAD3 staining in scr siRNA transfected CF and cytoplasmic SMAD3 staining in fibulin-6 siRNA transfected CF. A corresponding graph shows quantitative measurement of SMAD3 positive nuclei compared to the total number of cells (n = 3, p < 0.0001, nonparametric Mann-Whitney U test). The experiment was repeated three times with 10 pictures analyzed each time. Western blot showing (b) SMAD3 and (c) SMAD4 nuclear signal in TGF-β stimulated nuclear lysates compared to the non-stimulated set. Westerm blot and corresponding densitometric analysis show increased localization of (b) SMAD3 and (c) SMAD4 in nuclear extracts after TGF-β stimulation, which is absent in fibulin-6 KD CF (n = 5, p < 0.05 nonparametric Mann-Whitney U test).
