Figure 3.

Increased replication stress in 4N cells. A) Immunoblot analysis of total CHK1 and phospho-CHK1 levels in DLD-1 2N and 4N cells. GAPDH was used as protein loading control. B) Immunoblot showing increased levels of both RPA32 and phospho-RPA32 in DLD-1 4N cells. C) Analysis of phospho-CHK1 levels after addition of aphidicolin (0.2 µM) for 24 h to both sets of DLD-1 clones. GAPDH was used as protein loading control. D) Immunoblot analysis of total CHK1 and phospho-CHK1 in RKO 2N and 4N cells. GAPDH was used as protein loading control. E) Immunoblot showing increased levels of phospho-RPA32 vs. RPA32 in RKO tetraploid cells. GAPDH was used as protein loading control. F) Analysis of phospho-CHK1 levels after addition of aphidicolin (0.2 µM) for 24 h to both sets of RKO clones. GAPDH was used as protein loading control. G) Graph depicting percentage of growth impairment of 4N compared to 2N DLD-1 cells as measured by colony formation assay after treatment with ATRi (10 µM) for 24 h (n = 3/clone). H) Immunoblot analysis of total CHK1 and phospho-CHK1 levels in RPE1 wild-type (2N) and posttetraploid (4N) clones. GAPDH was used as protein loading control. I) Immunoblot showing increased levels of phospho-RPA32 vs. RPA32 in posttetraploid RPE1 cells. GAPDH was used as protein loading control. J, K) Plots showing time-course experiments to characterize delay in cell cycle. DLD-1 cells were pulse-labeled with BrdU and analyzed by FACS every 2 h for 12 h period. 4N cells exhibit slower progression through S phase (J) and slower rate going through G2 phase (K). L) Histogram showing mitotic timing of 2N and 4N cells quantified by live cell imaging in DLD-1 (n = 50 cells/clone). M) Histogram showing mitotic index of 2N and 4N DLD-1 cells based on DAPI imaging (n = 50 cells/clone). Statistical analysis was performed on trends using R package geepack for longitudinal data analysis. Data are reported as means ± sem. **P < 0.001, ***P < 0.0001 (n.s., not significant).