Fig. 3. Quantitative redistribution of Sp1, Sp3, ER and HDAC2 during mitosis. (A) MCF-7 cells were treated with 0.06 µg/ml colcemid for 16 hours and immunolabeled with anti-Sp1, anti-Sp3, anti-HDAC2 or anti-ER antibodies, while co-stained with DAPI, to identify the phase of the cell cycle. Cells were digitally imaged. Metaphase cells are recognized by the bright DAPI staining of their condensed chromatin. The pixel intensities of interphase and metaphase cells were quantified from the same image of one slide displaying both types of cells. The total intensities of 30-50 interphase cells and 30-50 metaphase cells were measured with AxioVision 4.1 software. Error bars show the standard deviation (s.d.). (B) The cell-cycle status of different cell populations was characterized by FACS analysis: MCF-7 cells grown in DMEM (DMEM), treated with 1.5 mM hydroxyurea (HU) or treated with 0.06 µg/ml colcemid (colcemid). (C) Immunoblots of cellular extracts of MCF-7 cells in different phases of the cell cycle as shown in (B) were probed with antibodies as indicated.