Fig 1: Massive genome re-replication is accompanied by altered replication dynamics.a HCT116 cells were treated with 250 nM MLN4924 for 24 h. Cell cycle changes were monitored by flow cytometry, and percentage of cells in each cell cycle stage is shown. See Supplementary Fig. 1 for gating strategy, schematic representation of cell distribution at the different cell cycle stages and additional time points and dose of MLN4924 treatment. Fluorophore Alexa 647 (APC) was used for EdU for all the flow cytometry experiments. Got similar results from 2 or more independent experiments. b Doxycycline inducible CDT1-GFP plasmid was stably transfected into U2OS cells (inducible CDT1-U2OS cells). Inducible CDT1-U2OS cells were cultured without (control) or with (CDT1 OE) doxycycline for 24 h (See Supplementary Fig. 3 for 48 h). c, d Changes in CDT1 levels (Fluorophore Alexa 488 (FITC) corresponding to cell cycle progression in HCT116 cells treated with 250 nM MLN4924 for 24 h are indicated by DAPI-DNA versus CDT1 (c) and by CDT1 versus EdU (d). e, f Changes in MCM2-pS139 levels (Fluorophore Alexa 488 (FITC) corresponding to cell cycle progression in HCT116 cells treated with 250 nM MLN4924 for 24 h are indicated by DAPI-DNA versus MCM2-pS139 (e) and by MCM2-pS139 versus EdU (f). g Replication profiles in HCT116 cells treated with 250 nM MLN4924 for 30 h measured by DNA combing. Cells were labeled with the thymidine analog IdU for 20 min (green), then CldU (red) for 20 min before collecting. Representative images of DNA combing for both the control and MLN4924-treated samples are shown (top). The replication fork progression rates (bottom left) and replication inter-origin distances (bottom right) are expressed as the median and interquartile range (total number of fibers counted are shown under the sample name). Mann–Whitney test was used for the statistical significance. p < 0.0001 by using the two-sided test for both fork rate and origin distance. Similar results were obtained from 3 independent experiments. h MLN4924 induced asymmetric replication forks in HCT116 cells. Cells were treated as in Fig. 1g. When the difference between the lengths of left forks and right forks emanating from the same origins was greater than 30%, these forks were classified as asymmetric forks. The percentages of asymmetric forks from each group are shown. The total number of forks analyzed are shown at the bottom right corner. Chi-square test was used for the statistical significance of the percentage difference of asymmetric fork between control and MLN4924 treated samples. Similar results were obtained from 2 more independent experiments.