FIGURE 4.

Knockdown of p53 expression in CHRF cells has little impact on cell cycle kinetics and does not affect spontaneous polyploidization in the absence of PMA treatment. Unstimulated CHRF-p53-A, CHRF-p53-B, and CHRF-neg-A cells were incubated for 12 h with 10μm BrdUrd and then stained intracellularly with an APC-conjugated anti-BrdUrd antibody to assess the amount of synthesized DNA and counterstained with PI to evaluate DNA content. The sum of 2 n/4 n cells gated by P₁ and 8 n/higher cells gated by P₂ represents the total number of cells undergoing DNA synthesis (BrdUrd⁺). Gates P₃ and P₄ represent the noncycling 2 n/4 n and 8 n or higher cells, respectively, and constitute the BrdUrd^– cells. Three independent biological experiments were carried out. Representative cell cycle/DNA synthesis (A) and ploidy among total cells (B) data are shown for CHRF-p53-A, CHRF-p53-B and CHRF-neg-A cells. In all three cases, only ∼0.7–0.8% of the total number of cells are shown to undergo spontaneous polyploidization. All data presented in this figure were acquired using protocol 2 for analysis of DNA synthesis as described under “Experimental Procedures.”