Apoptosis and cell-cycle analysis in preformed mouse polyploidy ESCs and their EB formation after expansion culture. Phosphohistone H3 (A) and caspase-3 (B) is shown in control (solvent-treated) E14 cells or in preformed polyploid (MNP) cells after cells were washed free of nocodazole or control solvent, recultured in complete medium containing LIF, and expanded by subculture for 4 passages (4-pass). They were then harvested, and multivariate cell-cycle analysis performed as in Figure 1. DNA content, percentage of polyploidy, and percentage of apoptosis are numerically indicated. (C) Apoptosis and polyploidy after 6 and 10 expansion passages of MNP is numerically shown. (D) Untreated control and MNP cells from panel A were then washed free of LIF and placed into EB medium for the indicated time, then harvested and apoptosis analysis done. DNA content and numerical percentages of polyploidy and apoptosis for untreated control cell–derived mEB cells and MNP-derived mEB cells are indicated. This experiment was repeated once with similar results.