Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels
1 From the Department of Molecular Genetics, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland OH, USA
2 The Department of Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA, USA
BMC Cell Biology 2006, 7:33 doi:10.1186/1471-2121-7-33Published: 30 August 2006
The appearance of a stimulated compared to an unstimulated cell, low power. Two NIH3T3 cultures were serum deprived for 30 hrs, microinjected with the PH-AKT-GFP plasmid, and cultured for 14 hrs prior to stimulation of only one of the cultures with 10% serum 10 min prior to photography. This movie is a sequence of fluorescent confocal images beginning near the coverslip and extending through one cell from the stimulated culture, and one cell from the unstimulated culture. The distribution of the fluorescence at the plasma membrane and away from the interior of the cell is apparent in the stimulated cell (40× objective, 10 micron steps).
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The appearance of a stimulated compared to an unstimulated cell, high power. An NIH3T3 culture was serum deprived, injected with the PH-AKT-GFP plasmid, and stimulated as above. Fluorescent confocal images (63X/1.4 objective) beginning at the coverslip and extending through the cell were taken of one cell 10 min following serum stimulation, and of another cell in an unstimulated culture prepared in parallel. The membrane structures observable following stimulation can be compared to the cytoplasmic localization in the unstimulated cell.
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GFP localization through a single, stimulated cell. An NIH3T3 culture was serum deprived, injected with the PH-AKT-GFP plasmid, and stimulated as above. This series of fluorescent confocal images (40X/1.25 objective) illustrates the appearance of cytoplasmic structures that are at times visible in these stimulated cultures.
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Additional file 4:
GSK3 activity in serum-deprived cultures. (A) NIH3T3 cells were synchronized by thymidine treatment and released for the indicated times prior to lysis and assay of the GSK3 activity. For comparison, NIH3T3 cells which had been deprived of serum for 48 hrs were analyzed for GSK3 activity without serum stimulation (0 hrs), and following serum stimulation for the indicated number of minutes. These are typical results of a single experiment. (B) To determine the effect of serum removal upon GSK3 activity, actively proliferating NIH3T3 cultures were deprived of serum for the indicated times prior to lysis and assay of GSK3 activity.
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