Research Paper Volume 11, Issue 23 pp 11686—11721

Figure 1. Most progeny coming from a Top2-mediated mitotic catastrophe is inviable. (A) Haploid TOP2 (WT) or top2-5 cells were grown at 25 °C and spread on YPD agar plates. Unbudded cells (G₁/G₀) were identified and photographed again after 6 h at 37 °C. Number of cell bodies (buds) coming from these G₁/G₀ cells were then counted and plotted as indicated. (B) The same analysis as in panel A but including data coming from independent experiments as well as after 24 h incubation at 37 °C (mean ± s.e.m., n=3). (C) The principle of the solid medium-based clonogenic assay. Unlike the liquid medium-based clonogenic assay, cells are spread on the Petri dish before the condition that challenges survivability is transiently triggered (Top2 inactivation in our study). In the solid medium-based assay, the colony forming unit (CFU) reading after the challenge is binary, irrespective of how far cells keep on dividing during the challenge: “0” if all clonal cells are inviable (grey); “1” if at least one cell from the clone stays viable (yellowish orange). (D) Time course of clonogenic survivability. Asynchronous top2-5 cultures growing at 25 °C were spread onto several YPD plates. The plates were incubated at 37 °C for different periods before transferring them 25 °C. Four days after the initial plating, visible colonies (macrocolonies) were counted and normalized to a control plate which was never incubated at 37 °C (0h). (E) Analysis of the origin of macrocolonies after the 6 h x 37 °C regime as determined after microscanning plates at the time of seeding (N=33 macrocolonies; 2:1 unbudded:budded ratio at seeding).