Fig. 3.

Asymmetric segregation of template DNA is modulated by environmental changes. (A) Bar graph showing a decreased frequency of asymmetric division of template DNA at lower cell densities, as well as a decreased frequency of asymmetric template DNA division under hypoxic conditions and serum deprivation. A549 and H441 cells are indicated by black and gray bars, respectively. Data are reported as mean ± SD from three independent experiments. *P < 0.0001, one-way ANOVA. Cell density is reported as million/cm², compared with 95% confluence. At least 250 anaphases were scored in each experiment. (B) Representative image of four-cell colonies derived from single A549 cells that were grown in BrdU, labeled with CTR (red), and cocultured with unlabeled A549 cells. Colonies contained all BrdU⁺ (green) cells (Upper), indicating symmetric divisions of the template DNA, or a mixture of BrdU⁺ and BrdU⁻ cells (Lower), indicating an asymmetric division. DNA was stained with DAPI (blue). The total number of colonies, colonies with all BrdU⁺ cells, and colonies with mixed BrdU⁺ and BrdU⁻ cells are shown. Numbers in parentheses represent the percentage of colonies with asymmetric divisions. The single cell–derived four-cell colonies comprise mixed BrdU⁺ and BrdU⁻ cells only when cocultured with neighboring A549 cells (no membrane barrier). When the colonies were separated from the cocultured cells by a membrane barrier, no asymmetric divisions were observed. (Scale bar: 20 μm.)