Microtubule disruption-induced mitotic arrest and polyploidy in mESCs. mESC lines E14 and R1 were treated with nocodazole (for microtubule depolymerization), paclitaxel (for microtubule overstabilization), or control solvent for 24 hours in complete culture medium containing LIF as described in “Materials and methods.” (A) Cells were harvested and assayed by multivariate permeabilized-cell cell-cycle analysis for simultaneous phospho(ser10)histone-H3 and DNA content. Regions 1 and 2 indicate E14 cells that are in M phase as indicated by increased phosphohistone-H3 content at 4C and 8C DNA content. (B) Results of polyploidy (cells with > 4C DNA content) analysis in E14 and R1 cells from 6 independent experiments is shown as the mean ± 1SD. (C) Percentage of M-phase cells (regions 1 and 2) are shown. (D) Relative frequency histograms of chromosome number in metaphase E14 cells treated with nocodazole for the indicated times showing an average of 40 chromosomes per cell (euploid) at 0 time and showing the increase in cells with 80 chromosomes (tetraploid) at 24 hours. Chromosome number indicates BIN number times 5. The experiment was repeated once with the E14 cell line and once with the R1 cell line with similar results. Chromosome counts in normal MEF cells are shown for comparison in Figure S2C. MEF cells had 40 chromosomes per cell. (E) Typical metaphase chromosome appearance in E14 cells before and after nocodazole treatment; the number of chromosomes is indicated (Nikon Labophot-2; 10 × 100; oil). (F) Wright-Giemsa stain of E14 cells harvested 24 hours after nocodazole treatment displaying a single nucleus. No E14 cells with more than 1 nucleus were observed (Nikon Labophot-2; 10 × 40).