Fibronectin1

E-cadherin (BD Transduction Labs, 610182; used for immunoblotting), E-cadherin (Zymed, 13-1900; used for immunofluorescence stainings), N-cadherin (Takara, M142), Zona Occludens-1 (Zymed, 617300), Paxillin (BD, 610052), Fibronectin1 (Sigma-Aldrich, F3648), Vimentin (Novus Biological, NB300–223), α-Tubulin (Sigma, T-9026), GAPDH (Abcam, ab9485), Alexa-Fluor 488 and 568 (Molecular Probes), secondary horse radish peroxidase (HRP)-conjugated antibodies against mouse and rabbit (Jackson ImmunoResearch), Phalloidin Alexa-Fluor 568 (Molecular Probes, A12380), 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9542), recombinant human TGFβ1 (R&D Systems, 240-B).

PC3-LN4 or BPH-1 cells (5×10⁵) were plated onto nanofiber scaffolds (Donaldson, Inc.) coated with 0.5 µg/cm² of 2∶1 (w/w) tenascin-C (Millipore):Fibronectin-1 (Sigma-Aldrich) or laminin (Sigma-Aldrich), respectively. The next day, cells were treated with s50-TBG-FeO-dextran (1.25 µg of Fe) in 1 ml of media. Treatment of cultures was staggered so that exposure times varied from 2 to 24 h. Cultures were fixed in 2% paraformaldehyde, developed with DAB-enhanced Prussian blue for iron and counterstained with Fast Red. Images were acquired using an Olympus BX60 fluorescent microscope 40× objective with a digital color Q Imaging Retiga 2000R Fast1394 camera as 8 bit RGB 1600×1200 pixels. Experiment was performed 4 times.