Fig. 5: Rio1 promotes the timely formation of structurally fit kinetochores.

a Real-time quantitative analysis of Ndc80-3GFP fluorescence levels at kinetochores in a RIO1-AID strain released from G1 in the presence of 500 µM auxin or a mock, and tracked through S-phase. The number of cells analysed (n) at each time-point derived from three independent biological experiments. The cells were imaged across different microscopy fields through 20–30 Z-planes, which were then vertically projected at maximum intensity to measure the fluorescence signals. A.U. arbitrary units. The data are plotted as mean ± SD, P-values were calculated with the unpaired, two-tailed Student’s t test. b Fluorescence levels of GFP-labelled kinetochore proteins at spindle-bound kinetochores in RIO1-AID strains treated with 500 µM auxin or a mock. The number of cells analysed (n) per cell cycle stage (G1, S-phase (S), metaphase (M), and anaphase (A)) derived from three independent biological experiments. The cells were imaged across different microscopy fields through 20–30 Z-planes, which were then vertically projected at maximum fluorescence intensity for signal measurement. A.U. arbitrary units. The data are plotted as mean ± SD. P-values were calculated with the unpaired, two-tailed Student’s t test. c Whole-cell levels of epitope-tagged Cse4, Mif2, Cnn1, and Ndc80 measured by western blot in RIO1-AID cells treated with auxin or a mock. OsTir1 or Pgk1 acted as the loading controls and internal references for relative quantifications (blots are shown in Supplementary Fig. 7a). The measurements were normalised to those quantitated in the mock-treated cells (value = 1). The singular data (white circles) derived from three independent biological experiments (n = 3) and are combined as mean ± SEM. P-values were calculated with the unpaired, two-tailed Student’s t test.