Fig. 4. Development of ef2-null (B,D,F) and parental Ax-2 cells (A,C,E) under submerged conditions. ef2-null and Ax-2 cells were harvested during the exponential growth phase, washed twice in BSS and plated in a 24-well titer plate at a density of 5x10⁵ cells/ml (1 ml of cell suspension/ well). This was followed by incubation at 22°C for 6.0 hours (A,B), for 7.5 hours (C,D) and 10.0 hours (E,F). At 6.0 hours of incubation, Ax-2 cells acquire aggregation competence (A), while ef2-null cells show no sign of cell aggregation (B). At 7.5 hours, aggregation streams are formed in Ax-2 cells (C), but not in ef2-null cells (D). At 10.0 hours, Ax-2 cells form tight aggregation streams (E), but ef2-null cells still remain as aggregation-competent cells (F). Similar results were obtained by observations in at least three independent experiments. Bars, 200 µm.