Fig. 4: CDK4/6-inhibition causes an origin licensing defect upstream of CDC6/CDT1 function, which can be sustained to trigger replication failure and aberrant mitosis in p53-deficient cells.

a–f Sequential CDK4/6 and licensing inhibition triggers mitosis with unreplicated DNA. a outline of the experiment. b QIBC analysis simultaneously monitoring DNA synthesis (EdU incorporation) and nuclear Cyclin A2 levels in single RPE CDC6d CDT1d cells 12 h after palbociclib release (>1000 cells per condition). Figure axes depict integrated fluorescence intensities (arb. units) per nucleus; Upon release cells were either mock treated (grey) and exposed to ADN treatment (blue); dotted line box highlights mid S-phase cells (classified based on CyclinA2). c violin plots depict distribution of EdU signals in three independent mid S-phase populations per condition, treated and classified as in (a, b) (n = 300 mid-S phase cells per replicate). Asterisk indicates a significant disparity between population means; n = 3 technical replicates per condition, p = 0.00002 (two-tailed paired t test). d mitotic phenotypes upon treatment described in (a); compound DAPI images show representative mitotic nuclei; scale bars, 10 μm; bar graph depicts quantification of the percentage of anaphase nuclei among mitotic cells; bars indicate mean % of anaphase-like nuclei; error bars indicate SD of four independent replicates. Asterisk indicates p = 0.0002 (two-tailed paired t test). e Immunoblots examining the chromatin occupancy of indicated proteins and/or phosphorylated epitopes upon treatment described in (a); the reduction of PCNA as well as MCM4 and MCM2 serine 53 phosphoryation confirm a licensing defect. β-actin served as loading control. Representative blots are shown, with the experiment repeated twice independently with consistent results. f Top panel depicts outline of the experiment and bar graph below shows mean integrated intensity of nuclear EdU signals for each condition (arb. units); error bars indicate SEM of three independent experiments; black dots indicate replicate means. Asterisk indicates a significant disparity between population means; p = 0.0004 (two-tailed paired t test). Source data are provided as a Source Data file.