Tetraploid (4N) DLD-1 clones were generated and characterized as described previously [32 (link)]. Briefly, diploid (2N) cells were treated with 1.5 g/mL dihydrocytochalasin B (DCB) (Sigma Aldrich, St. Louis, MO, USA) for 20 h, and limiting dilution in 96-well plates was used to isolate and expand the 4N clones from single cells. Metaphase spreads were collected from 4N clones, and chromosome counting was performed to confirm ploidy/DNA content. G2-synchronized cells (9 M RO-3306 for 18 h) were fixed with ice-cold methanol for 10 min and 5 M Cell Tracker Green CMFDA Dye (Thermo Fisher Scientific), and 300 nM DAPI was used to label the cytoplasm and nucleus, respectively, of each cell. Z-stack images spanning the entire height of individual cells were acquired with 0.6 µm steps using a swept field confocal system on a Nikon Eclipse TE2000-U (Nikon Instruments Inc., Melville, NY, USA) inverted microscope equipped with a 60× objective. Cell and nuclear volume analyses were performed in FIJI using a macro for three-dimensional reconstruction [35 (link)]. From the diploid cells (2N), two small tetraploid clonal lines (S1 and S2) and two large tetraploid clonal lines (L1 and L2) were generated and used throughout the experiment. The cell and nuclear radii were calculated from volume measurements assuming a spherical shape when in suspension.
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