Depletion of Cik1-Kar3 leads to chromosome mis-segregation and has phenotypes similar to those of knockouts. (A) Schematic overview of Cik1-Kar3 protein structure. Functionally important regions are highlighted and assigned accordingly. (B) Western blot analysis of Cik1-AID and Kar3-AID upon addition of NAA. Kar3 is present in excess over Cik1 in mitotic cells. α-Factor–arrested cells have increased amounts of a shorter Cik1 isoform. Long exp., ong exposure; α-Tub, anti-Tubulin blot. (C) Serial dilution spot assays compare cik1 and kar3 genetic deletions with the respective conditional depletion alleles. Temperature sensitivity was assayed at 37°C. Plates marked +NAA contained 1 mM of NAA. Plates were incubated for 2.5 d at the stated temperature. (D) Analysis of Cik1 or Kar3 depletion in a checkpoint-deficient strain. Plates were incubated for 2.5 d at 30°C. (E) Schematic representation of mitotic timing. Metaphase duration is defined as the time between SPB separation and anaphase onset. Gallery of consecutive frames from live-cell imaging of Cik1-AID strains. aux, auxin. The SPB was labeled with Spc42-RedStar2, kinetochores with Ndc80-GFP. Scale bar is 2 µm. Movies are aligned with the separation of SPBs displayed in the first frame. Quantification of metaphase duration in individual Cik1-AID cells. A total of 200 cells from two biological replicates were analyzed. Open circles represent mean duration from each individual experiment; error bars show 95% CI. χ² test was applied; *, P < 0.0001. (F) Analysis of spindle size and dynamics in Cik1-depleted cells. Left panel: Gallery of consecutive frames from live-cell imaging. Middle and right panels: Quantification of spindle elongation for n = 15 cells. Error bars are means with 95% CIs. Student’s two-tailed t tests were made (*, P < 0.0001; NS, P = 0.59).