Apoptosis and cell-cycle assays. (A) Apoptosis assay. Annexin V–positive cells were quantified by flow cytometry at days 2 and 5 after removal of doxycycline. The top panel shows a representative result of FOXO3–re-expressing cells at day 5. More apoptotic cells were observed under conditions with doxycycline treatment than those without doxycycline treatment. The bottom panel indicates the proportion of annexin V–positive cells at days 2 and 5. FOXO3 re-expression increased the amount of apoptotic cells at day 5, whereas no clear changes were observed in PRDM1–re-expressing cells at days 2 or 5. Experiments were performed in triplicate and standard deviations and average scores are shown. Dox indicates doxycycline. *Significantly different Dox(−) and Dox(+) samples (P < .05). (B) Cell-cycle assay assays conducted using PI at days 2 and 5. The top panel shows a representative result of FOXO3 re-expressing cells at day 2. The proportion of S-phase cells was lower under conditions without doxycycline treatment than those with doxycycline treatment. The bottom panel shows the proportion of S-phase cells at days 2 and 5. FOXO3 re-expression decreased the proportion of S-phase cells at days 2 and 5, whereas a decrease in the proportion of S-phase cells only occurred at day 5 with PRDM1 re-expression. Experiments were performed in triplicate and standard deviations and average scores are shown. *Significantly different Dox(−) and Dox(+) samples (P < .05).