Analysis of apoptosis of E14 cells before and after treatment with microtubule-disrupting agents or after DNA damage. (A) E14 mESCs were treated with the indicated agent for 24 hours and harvested as in Figure 1. Cells were analyzed by permeabilized-cell flow cytometry as in Figure 1 except an antibody to activated caspase-3 was used. Cells above the bar are positive and those below the bar are negative for caspase-3 activation. (B) Caspase-3 activation in day-3 mEB cells after 24 hours of treatment. Percentage apoptosis (mean ± 1SD for 3 independent experiments) as indicated by caspase-3 activation. (C) Percentage apoptosis (mean ± 1SD for 3 independent experiments) as indicated by sub-G₁ cells. *Statistically significant difference from control; P < .05. (D-E) Apoptosis measurement in treated and untreated mESCs or mEB cells as indicated by Annexin-V binding. Cells were simultaneously stained with propidium iodide to indicate cellular membrane integrity. Early apoptosis (Annexin-V⁺ and PI⁺) and total apoptosis (Annexin-V⁺ and PI^+/−) for E14 or mEB cells before and after treatment as in panels A and B. Results are mean percentages ± 1SD for 3 independent experiments.