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First published online 21 February 2006
doi: 10.1242/jcs.02829

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Sp1 and Sp3 foci distribution throughout mitosis

Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0V9, Canada

View larger version (96K): [in a new window]   Fig. 1. Temporal distribution of transcription factors Sp1 and Sp3 during mitosis. MCF-7 cells were subjected to indirect immunofluorescence labeling with an antibody against Sp1 or Sp3, and co-stained with DAPI for identification of cell-cycle stage. Cells at various phases of mitosis were digitally imaged. Bar, 5 µm.

View larger version (85K): [in a new window]   Fig. 2. Temporal distribution of ER and HDAC2 during mitosis. MCF-7 cells were immunolabeled with antibodies against ER or HDAC2, and co-stained with DAPI. Cells at various phases of mitosis were digitally imaged. Bar, 5 µm.

View larger version (27K): [in a new window]   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.

View larger version (21K): [in a new window]   Fig. 4. Equal partitioning of Sp1 and Sp3 between daughter nuclei. MCF-7 cells grown on coverslips were treated with 0.06 µg/ml colcemid for 16 hours, cultured for 3 more hours in fresh medium and immunolabeled with anti-Sp1 and anti-Sp3 while co-stained with DAPI. Bar, 5 µm. A quantitative image analysis was performed to determine the relative levels of Sp1 and Sp3 in the telophase cells. The partition coefficient (PC) indicates the ratio of integrated signal intensities between two daughter nuclei. Student's t-test was applied to assess the significance of observed differences.

View larger version (66K): [in a new window]   Fig. 5. Distribution of Sp1 and Sp3 throughout the mitosis stages. MCF-7 cells were grown on coverslips in estrogen-complete medium, fixed, and double labeled with anti-Sp1 and anti-Sp3 antibodies. DNA was stained by DAPI. Sp1 and Sp3 distributions were visualized by fluorescence microscopy and image deconvolution as described in Materials and Methods. Single optical sections are shown. Yellow in the merged images signifies colocalization. Bars, 5 µm.

View larger version (77K): [in a new window]   Fig. 6. Temporal mitotic distribution of Sp3 and lamins. MCF-7 cells were grown on coverslips in estrogen-complete medium, fixed, and double labeled with rabbit anti-Sp3 antibodies and mouse monoclonal anti-lamin A/C antibodies. DNA was stained by DAPI. Spatial distribution was visualized by fluorescence microscopy and image deconvolution as described in Materials and Methods. Single optical sections are shown. Bar, 5 µm.

View larger version (101K): [in a new window]   Fig. 7. Sequential re-entry of Sp1, Sp3 and RNAP II into daughter nuclei. MCF-7 cells were grown on coverslips in estrogen-complete medium, fixed, and double labeled with anti-Sp1 or anti-Sp3 and anti-RNAP II antibodies or with anti-Sp1 and anti-Sp3 antibodies. DNA was stained by DAPI. Spatial distribution was visualized by fluorescence microscopy in the ApoTome mode. Bars, 5 µm.

View larger version (124K): [in a new window]   Fig. 8. Mitotic colocalization of Sp1 with F-actin. MCF-7 cells were grown on coverslips in estrogen-complete medium, fixed, and labeled with rabbit polyclonal anti-Sp1 antibodies and mouse monoclonal anti-calnexin or anti-cytokeratin 8 or anti-cytokeratin 18 antibodies. F-actin was labeled by Alexa-Fluor-488-conjugated phalloidin. DNA was stained by DAPI. Spatial distribution was visualized by fluorescence microscopy and image deconvolution as described in Materials and Methods. Single optical sections are shown. Yellow in the merge images signifies colocalization. Bar, 5 µm.

View larger version (51K): [in a new window]   Fig. 9. Co-sedimentation of Sp1 and Sp3 with F-actin. MCF-7 nuclear extract was mixed with G-buffer and polymerization inducer (lanes 1 and 2), G-actin (lanes 3 and 4) or F-actin (lanes 5 and 6). BSA was added as negative control. Mixtures were spun. Pellet (lanes 1, 3 and 5) and supernatant (lanes 2, 4 and 6) fractions were resolved by SDS-PAGE. Sp1 and Sp3 were detected by western blot (WB), whereas BSA and actin were visualized by Coomassie Blue (CB) staining