Overexpression of DEAF-1 in MCF10A cells leads to enhanced cellular proliferation within acini. A) MCF10A-EcoR cells transduced with either a Deaf-1-expressing or empty pBabe-puro retrovirus were analyzed by western blotting of whole cell lysates using anti-DEAF-1 polyclonal antisera. Blotting for tubulin provided a loading control. B) Control or Deaf-1 expressing MCF10-EcoR cells were plated at 4,000 cells/well in eight-well glass chamber slides. Wells were pre-coated with Matrigel and the final culture medium contained 20 ng/ml EGF and 2% Matrigel. Acini were fixed in 2% paraformaldehyde after 8 days in culture, immunostained with anti-DEAF-1 (green), counterstained with DAPI (blue) and acini visualized by confocal microscopy (DAPI = uv; and DEAF-1 = 488 nm). Scale bar represents 47.6 μm. C) Transduced acini were grown as in B) from control MCF10A-EcoR cells, DEAF-1-transduced-MCF10A cells, and malignant MCF10CA1h cells. Cells were fixed in 2% PFA after 6, 8 and 10 days in culture. Acini were immunostained with anti-Ki67 (green) and counterstained with DAPI (blue) following 6, 8 and 10 days in culture. At least three independent experiments were performed. Acini were visualized with a confocal microscope (DAPI = uv; and Ki67 = 488 nm). Scale bar represents 47.6 μm.