Extended Data Fig. 9: Mitotic delays induced by CENP-E inhibition trigger G1 arrest.
From: MDM2 functions as a timer reporting the length of mitosis
a,b, The amount of MDM2 was measured by Western blotting in asynchronous (AS) or mitotic hTERT-RPE1 p53WT cells, arrested in mitosis for 18 h with 100 ng/ml nocodazole (N), 5 µM S-trityl L-cysteine (S), or 300 nM CENP-E inhibitor (C). Relative MDM2 levels across the different conditions were quantified for two distinct MDM2 antibodies (b) (mean ± s.d.; n = 3 independent experiments; MDM2 IF2, p = 2.5E−8 AS vs. N, p = 2.7E−8 AS vs. S), p = 5.2E−8 AS vs. C; MDM2 SMP14, p = 1.4E−7 AS vs. N, p = 1.1E−7 AS vs. S, p = 4.0E−7 AS vs. C). c, Western blot of cyclin B1 positive mitotic (M) and cyclin B1 negative (G1) hTERT-RPE1 p53WT cells arrested for either 1 h or 4 h in mitosis with 30 nM CENPE-inhibitor (CENPEi), in the absence (–) or presence (+) of 100 nM MD-224 for the final hour. 3 independent experiments showed similar results. d, Representative images of hTERT-RPE1 p53WT passing from mitosis into the following cell cycle after arrest in mitosis for 45 min with 30 nM CENP-E inhibitor (CENP-EiShort) in the absence or presence of 100 nM MD-224 prior to washout. The mitotic cells were tracked after washout and then imaged. e,f, Single cell traces of hTERT-RPE1 p53WT FUCCI cells treated as in d passing from mitosis into the following cell cycle. For each condition, the number of individual daughter cells analysed are indicated. The percentage of cells undergoing the G1/S transition is plotted (mean ± s.d.), and the duration of G1 in those cells that entered the next S-phase is shown in a box and whiskers plot (median, 25th and 75th percentiles, and whiskers extending to minimum and maximum values) with the mean (+) for the different conditions; n = 3 independent experiments per condition; p = 5.0E−3 G1/S transition Control vs. +MD-224, p = 3.1E−7 G1 length in cells entering S phase Control vs. +MD-224) (d–f). Data were analysed using one-way ANOVA test with Tukey’s multiple comparisons (b) or unpaired two-tailed t-test with Welch’s correction (f) (ns, not significant; **, p < 0.01; ****, p < 0.0001). Source numerical data and unprocessed blots are available in source data.
