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Fig. 2. Simultaneous deregulation of Cdt1+CDC6 or Cdt1+ORC1 induces re-replication, and triple overexpression synergistically induces strongest re-replication in HEK 293T cells. Cells were transfected with mixtures of the three different expression vectors (T7-Cdt1, Flag-ORC1, 2HA-CDC6) or their empty vectors, as indicated. In all experiments, a GFP expression vector was also included to monitor transfection efficiency. Forty-eight hours after transfection, cells were analyzed. (A,B) DNA content was analyzed by flow cytometry. In these diagrams, the x-axis is FL2-A (area) and represents whole propidium iodide (PI) signals and thus DNA content. The y-axis is FL2-W (width) and represents the duration of PI signals. Dots with higher FL2-W signals, which result from aggregated cells or cell debris, were excluded from measurement of re-replicated cells. The means and SDs of the percentages of re-replicated cells (the DNA content higher than 4N) from two independent experiments are shown in B. (C) Whole-cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. GFP served as a control. The signal intensities of the phospho-ATM and phospho-Chk2 proteins were quantified and the means and SDs from two independent experiments are shown with the Cdt1 overexpression alone set at 100 (lower panel). (D) Whole-cell extracts or chromatin- and nuclear-matrix-bound fractions were prepared and immunoblotted with the indicated antibodies. The signal intensities of the chromatin-bound MCM3 and MCM7 proteins were quantified, normalized to the signals of nuclear lamin C, and shown with the value of control transfectant set at 100. The means and SDs from two independent experiments are given. *P<0.05.
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