1. Log T = 0 T = 2.5 T = 5 T = 7.5 T = 10 T = 12.5 T = 15 T = 17.5 T = 20
Supplemental Figure 1. FACS analysis of synchronized HeLa cells. HeLa cells were arrested at the G1/S border by treatment with thymidine followed by mimosine as described in Materials and Methods. Cells were released into fresh media and at 2.5 hour time points, 5 x 105 cells were fixed with ethanol, stained with propidium iodide and analyzed by FACs. Asynchronous (Log) cells and the time in hours after release is displayed in the upper right corner of each graph. The x-axis represents DNA content and the y-axis represents the number of cells counted. Extrapolation of the peak in the Log phase cells indicates that an x-value of 320 represents 2N; therefore a value of 640 would represent 4N. Prior to release (T = 0), the majority of cells were sufficiently arrested at the G1/S border as demonstrated by the single peak. After release, the cells rapidly entered S-phase with most of the cells becoming 4N by 10 hours, as demonstrated by the shift in the peak to the right. Based on this, we assigned 2.5 hours as early S-phase, 5 hours as mid S-phase and 7.5 hours as late S-phase. At 10 hours, the majority of the cells were 4N indicating completion of S-phase. This time point represents G2 since there is no apparent increase in 2N cells. At 12.5 hours, approximately half of the cells divided as indicated by the increase in 2N cells indicating that the remaining 4N cells were in mitosis. By 15 hours, the majority of cells had exited mitosis and returned to G1-phase.