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. 2021 Aug 9;12:4789. doi: 10.1038/s41467-021-24841-y

Fig. 6. RIT1M90I weakens the spindle assembly checkpoint.

Fig. 6

a Western blot of RIT1 expression in parental and RIT1M90I-expressing HeLa H2B-GFP cells. Vinculin was used as a loading control. b Duration of mitosis was measured as time from nuclear envelope breakdown (NEBD) to the onset of anaphase. Each frame represents movie stills from time-lapse live-cell imaging of parental HeLa H2B-GFP cells undergoing mitosis, scale bar = 5 µm. c Time-lapse fluorescence microscopy of time in mitosis in asynchronous parental and RIT1M90I-expressing HeLa H2B-GFP cells in normal media conditions (control) or treated with 0.5 µM reversine (Rev) two hours before imaging. n = 50 cells per condition. Mitotic timing was measured from time of NEBD to anaphase onset. d Alternative representation of data from (c). Data shown are the mean and individual data points of each condition. ****p = 9.06e−9, n.s., p = 0.1754 by unpaired two-tailed t-test. e Mitotic index calculated as the percentage of mitotic cells in a frame at a chosen time point. Box plots show the median (center line), first and third quartiles (box edges), and the min and max range (whiskers). n = 2 biological replicates, 6 time points per condition, n.s., p = 0.3587 by unpaired two-tailed t-test. f Comparison of mitotic error rates in HeLa H2B-GFP cells expressing RIT1M90I compared to parental HeLa H2B-GFP cells in vehicle (DMSO)-treated and alisertib-treated (1 μM) conditions. n = 3 biological replicates, error bars indicate s.d., ***p = 0.0004, **p = 0.0029 by unpaired two-tailed t-test. g Representative images of mitotic errors from HeLa cells expressing RIT1M90I and quantified in (f), scale bar = 5 µm. h Time-lapse fluorescence microscopy of time in mitosis in asynchronous parental and RIT1M90I-expressing HeLa H2B-GFP cells in normal media conditions   or treated with 1 µM alisertib two hours before imaging. n = 50 cells per condition. i Proposed model explaining enhanced efficacy of Aurora kinase inhibitors in RIT1M90I-mutant cells. Source data are provided as a Source Data file.