Extended Data Fig. 11: Polyploidization upon oncogene overexpression.
From: Multigenerational cell tracking of DNA replication and heritable DNA damage
(a) High-content microscopy derived cell cycle profiles of 53BP1-PCNA U-2 OS cells overexpressing either HRAS or Cyclin E1 that were treated or not with 4 Gy of IR. The percentage of hyperploid cells (> 4 N DNA content, marked by the dashed vertical line) is indicated. (b) Representative images of 53BP1-PCNA U-2 OS cells overexpressing HRAS with increased ploidy (2 N, 4 N, 8 N). Scale bar, 10 µm. (c) Bar chart depicting the percentage of polyploid cells overexpressing either HRAS or Cyclin E1 that were treated or not with 4 Gy of IR. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post hoc test. (d) Cell cycle resolved EdU profiles of 53BP1-PCNA U-2 OS cells overexpressing HRAS or Cyclin E1. Cells were either untreated or treated with 4 Gy of IR. (e) Video stills of 53BP1-PCNA U-2 OS cells overexpressing HRAS that are maintaining polyploidy through multiple cell generations. The polyploid granddaughter cells with 8 N DNA content are identified in a scatter plot of mean vs total DAPI intensity post live imaging. (f) Representative single cell lineages from live imaging of 53BP1-PCNA U-2 OS cells that overexpress HRAS. Lineages depict endo-replication and re-replication, respectively. (g) Western blot validation of inducible expression of Cyclin E1 in 53BP1-PCNA RPE-1 cells. Cells were induced with Doxycycline (100 ng/ml) for 24 h. (h) High-content microscopy derived cell cycle profiles of 53BP1-PCNA RPE-1 with inducible expression of Cyclin E1. The percentage of hyperploid cells (> 4 N DNA content, marked by the dashed red vertical line) is indicated. Scale bars, 10 µm. For gel source data, see Supplementary Fig. 1.
