Cdc14 oscillations fulfill the kinase requirement of the two-hit model. (a) GAL-CLB2dBΔ cfi1-6Cdk (Ry1545) cells were arrested with α-factor in YEPR and released into medium supplemented with 1% galactose to induce the expression of Clb2. The percentages of cells with metaphase spindles (closed squares), anaphase spindles (closed circles), and cells with Cdc14 released from the nucleolus (open circles) were determined at the indicated times. (b and c) GAL-CLB2dBΔ cfi1-6Cdk cdc15-as1 (Ry1715) cells were arrested with α-factor in YEPR and released into medium supplemented with 1% galactose. 195 min into the release (b) or at the moment of the release (c), 5 µM 1NM-PP1 analogue 9 inhibitor (Bishop et al., 2001) was added to inactivate the cdc15-as1 allele. Cell samples were analyzed as in a. (d and e) GAL-CLB2dBΔ (Ry430) cells were arrested in G1 with α-factor in YEPR and released into fresh medium supplemented with 1 (d) or 0.05% (e) galactose. Cell samples were analyzed as in a. (f and g) GAL-CLB2dBΔ cdc5-1 (Ry1393) cells were treated as in d and e, respectively. Cells were shifted at the restrictive temperature 165 min after the release. Cell samples were analyzed as in a. Arrows indicate the time of inhibitor addition in b (+In) and the shift to the restrictive temperature in f and g.