Fig. 5: Oncogene overexpression induces two distinct routes towards polyploidization.

a, Single-cell lineage from 53BP1–PCNA U-2 OS cells overexpressing HRAS. PCNA foci, 53BP1 foci and area of the nucleus over 70 h of live imaging are depicted. Sequential staining intensities from individual cells of the lineage and mean intensities from the corresponding reference populations are depicted for the markers pRb, γH2AX, p21 and p53. The total DAPI intensity per nucleus from the corresponding reference populations is depicted as well as from the single cells at the end of live imaging. Representative images of the polyploid cells are included as well as their position within the DAPI scatter plot of the G1 and G2 populations. Scale bars, 10 µm. The box plots show the IQR (box limits), with median (centre line) and the whiskers define the lower and upper adjacent value; dots show outliers greater than Q3 + 1.5 × IQR. Avg., average. b, t-Distributed stochastic neighbour embedding analysis of scRNA-seq results. 2N–4N, cells with 2N–4N DNA content; polyploid, cells with a DNA content >4N; pevo., pevonedistat-treated; HRAS, HRAS-overexpressing cells. n > 300 cells per condition. c, Subclustering of the HRAS polyploid sample with a Louvain resolution of 0.5. d, GO analysis of the top 28 genes from the overlap between HRAS polyploid and pevonedistat polyploid subclusters. FDR < 0.00001, P < 0.00001, FC > 4. e, STRING functional protein association network analysis of the top 28 genes. f, Cell-cycle-resolved nuclear cyclin A levels in U-2 OS cells either untreated or treated with 175 nM of pevonedistat for 24 h and then released into fresh medium for 42 h. g, The cellular behaviour towards polyploidy from live imaging experiments of 53BP1–PCNA U-2 OS cells treated with 175 nM of pevonedistat, 20 μM of etoposide or 5 μM of RO3306 for 24 h and then released for 42 h. Mean and individual values are depicted from two biological replicates based on live imaging data.

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