Fig. 5: Defining the MDM2 threshold in mitosis for p21 induction and robust cell-cycle arrest in G1.

a, Single cell traces of hTERT-RPE1 p53^WT and p53^KO FUCCI cells passing from mitosis into the following cell cycle after arrest in mitosis (M) for either 30 min (Noc^Short) or 4 h (Noc^Long) with 25 ng ml⁻¹ nocodazole, in the absence or presence of 100 nM MD-224 for 30 min before washout. The mitotic cells were tracked after washout and imaged up to 60 h post mitosis (n = 3 independent experiments per condition). b, Mitotic hTERT-RPE1 p53^WT FUCCI cells in asynchronous culture (AS^{No delay}) were treated for 30 min with DMSO control, MD-224 or Nutlin-3a, the drugs washed out and the cells then tracked into the following cell cycle (n = 3 independent experiments per condition). c, MPS1 inhibitor was used to override the Noc^Short spindle assembly checkpoint arrest. Single cell traces of hTERT-RPE1 p53^WT FUCCI cells in the Noc^Short + MPS1i condition passing from mitosis into the following cell cycle (n = 3 independent experiments). a–c, For each condition, the number of individual daughter cells analysed are indicated. For cells that did not arrest in G1 and entered the next S phase, the mean length of G1 along with the number of cells undergoing the G1/S transition are indicated above each panel of traces (mean ± s.d.). The dotted line in each panel indicates the mean length of G1. Box and whiskers plots for G1 length show the median, the 25th and 75th percentiles, whiskers extending to minimum and maximum values, and the mean (+). d, The percentage of cells arrested in G1 is plotted (mean ± s.d.; n = 3 independent experiments per condition). e, Single cell traces of hTERT-RPE1 p53^WT FUCCI cells in either G1/S or S/G2 in asynchronous cultures were treated with MD-224 for 30 min as in the Noc^Short condition, washed to remove the drug, then tracked into the following cell cycle. The proportion of cells arresting in G1 and entering the next S phase are shown (n = 3 independent experiments; P = 6.2 × 10⁻⁹ p53^WT Noc^Short versus p53^KO Noc^Short, P = 6.5 × 10⁻⁷ p53^WT Noc^Short versus p53^WT Noc^Long, P = 2.5 × 10⁻¹³ p53^WT Noc^Long versus p53^KO Noc^Long, P = 2.1 × 10⁻⁷ p53^WT Noc^Short versus p53^WT Noc^Short + MD-224, P = 1.1 × 10⁻¹³ p53^WT Noc^Short + MD-224 versus p53^KO Noc^Short + MD-224, P = 7.8 × 10⁻⁴ p53^WT Noc^Short versus p53^WT Noc^Short + MPS1i, P = 3.4 × 10⁻¹⁰ control versus (+)MD-224, P = 0.99 control versus (+)Nutlin-3a). f, Levels of p21 in single hTERT-RPE1 p53^WT p21–GFP cells were followed post mitosis in new G1 cells for the Noc^Long (n = 33 cells), Noc^Short (n = 39 cells) and Noc^Short + MD-224 (n = 36 cells) conditions (mean ± s.e.m.; n = 3 independent experiments per condition). g, Titration of MD-224 to reveal the threshold concentration required for G1 arrest (black line) of hTERT-RPE1 p53^WT FUCCI cells. MDM2 levels were determined by western blotting (mean ± s.e.m.; n = 4 independent experiments) and p21 levels (mean, red) and proportion of cells arrested in G1 (black) by immunofluorescence microscopy (mean ± s.e.m.; n = 3 independent experiments). The data were analysed using a one-way ANOVA test with Tukey’s multiple comparisons for d (***P < 0.001, ****P < 0.0001).

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