Supplementary Figure 1: Identification and characterization of Cyclin A2-Venus RPE1 clones.

(a) Genomic PCR screen for correctly targeted Cyclin A2-Venus integrants following flow cytometry selection. Primer sets consisted of an oligonucleotide annealing outside the targeting construct (either 5′ (#1) or 3′ (#4)) and oligonucleotides annealing to the ORF of Venus (2 or 3). (b) PCR analysis with different primer combinations of a positive clone compared with parental RPE1 cells. (c) Single-cell Cyclin A2-Venus destruction assays of unsynchronised RPE1 Cyclin A2-Venus cells (n = 8 cells). Error bars indicate s.d. and are representative of two independent experiments. (d) Single-cell Cyclin A2 destruction assays of asynchronously growing RPE1 Cyclin A2-Venus cells treated with DMA (10 μM) and MPS1 inhibitor reversine (0.5 μM) or Mad2 siRNA (20 μM) for 48 hours (n = 6 cells for each condition). Images in c) and d) were acquired at 3 minutes intervals and the total cell fluorescence was quantified. Fluorescence intensities were normalized to the level at nuclear envelope breakdown (NEBD) and aligned at metaphase and NEBD respectively. Error bars indicate s.d. and are representative of two independent experiments. (e) Scatter plot of the time between nuclear envelope breakdown (NEBD) and exit from mitosis as judged by the disappearance of the Cyclin B1-Venus signal in Cyclin B1-Venus cells were treated simultaneously with DMA (10μM) and the indicated concentration of AZ3146. The black line in each condition represents the mean. n = 50 cells per condition. Representative of three independent experiments. Scale bar = 10 μm.