Fig. 4. Cells depleted of UbcH10 degrade cyclin A correctly in mitosis but not in the following interphase. (A) Cells depleted of UbcH10 degrade cyclin A with normal kinetics. HeLa cells treated with 40 nM of GAPDH (control) or UbcH10 siRNA were injected in late G2 phase with a plasmid encoding cyclin A tagged with Venus (5 ng/µl) and followed by time-lapse fluorescence and DIC microscopy. Images were taken every 3 minutes (exposure 200 mseconds), and the total fluorescence minus background was measured for each cell in successive images of a time series and plotted against time. The degradation curves shown are representative of nine control cells and 16 UbcH10 siRNA-treated cells in three independent experiments. Cells expressing low and high levels of cyclin-A–GFP were analysed to exclude any effects caused by overexpressing cyclin A. Four control cells expressing similar levels of cyclin A to four UbcH10-depleted cells are shown. Time 0 represents time of nuclear envelope breakdown. (B) Depleting UbcH10 causes cells to prematurely begin DNA synthesis, which requires cyclin A. hTERT-RPE cells were transfected with 40 nM control siRNA (GAPDH), and siRNA targeting UbcH10 or UbcH10+CycA, and synchronised in G0 by serum starvation for 24 hours. Cells were stimulated to re-enter the cell cycle by adding serum and harvested at the indicated time points. (i) Progression through the cell cycle was assayed by propidium iodide staining and flow cytometry analysis. (ii) Incorporation of BrdU that had been added 30 minutes before each time point. (iii) Protein levels were assayed by immunoblotting with the indicated antibodies. C, asynchronous cells. Results are representative of three independent experiments.