Fig. 8: RioK1 ensures correct cenRNA levels, faithful kinetochore assembly, and chromosome stability in human cells.

a Left: representative immunofluorescence (IF) images of RioK1 at kinetochores (CREST) in prometaphase and metaphase cells. Right: RioK1 levels at kinetochores (versus CREST) through the cell cycle. The numbers of cells analysed (n) derived from three replicate experiments. The data are shown as mean ± SD. P-values were calculated with the unpaired, two-tailed Student’s t test. Supplementary Fig. 8a shows IF images of RioK1 through the cell cycle. b Over-time levels of mAID-RioK1 and GAPDH (loading control) in mAID-RIOK1 cells treated with 500 µM auxin. c Cell cycle analysis (FACS) of mAID-RIOK1 cells treated with 500 µM auxin or a mock at mitotic entry. Cells were probed after 3 h or 24 h of treatment (four (n = 4) and five (n = 5) replicates per condition, respectively, Supplementary Fig. 8c). The data are shown as mean ± SD, P-values were calculated with the unpaired, two-tailed Student’s t test. d, Cen11 RNA levels (RT-qPCR) in mAID-RIOK1 cells treated for 3 h with auxin or a mock (five replicative experiments, n = 5) were normalised to GAPDH mRNA levels, and then referenced to the cen11 RNA levels in the mock-treated cells. The data are shown as mean ± SEM. P-values were calculated with the unpaired, two-tailed Student’s t test. mAID-RioK1 and GAPDH levels before and after auxin treatment are shown. e CENP-A, Ndc80, and Aurora B levels at kinetochores formed de novo without RioK1 (Supplementary Fig. 8b). Protein levels were quantified by IF analysis and normalised to DNA (DAPI) levels. Protein:DAPI ratios are shown as SuperPlots. The number of cells analysed (n) derived from six (CENP-A), seven (Ndc80), or three (Aurora B) replicate experiments. P-values were calculated with the paired, two-tailed Student’s t test. f Cells (%) containing micronuclei formed during mitosis in RIOK1 and mAID-RIOK1 cells treated at mitotic entry with a mock or 500 µM auxin (3 h). The number of cells analysed (3387 and 3461; respectively) derived from six experiments (n = 6). The data are shown as mean ± SD. P-values were calculated with the unpaired, two-tailed Student’s t test. Right: representative images of DAPI-stained cells containing (orange) or lacking (blue) mAID-RioK1 activity. The white arrowheads indicate micronuclei.