Fig. 4. Drosophila Gie is required for chromosome segregation. (A) Reduction of dGie mRNA levels by RNAi. Drosophila S2 cells were cultured with the control dsRNA (left) or dGie dsRNA (right) for 4 days, and the level of dGie transcript was measured by semiquantitative reverse-transcription PCR with decreasing amounts of the templates. Aurora-B mRNA was used as a loading control. (B) Flow cytometry analyses of the control (left) and dGie-dsRNA-treated (right) S2 cells. (C) The control (top) and dsRNA-treated (middle and bottom) S2 cells were stained with PicoGreen and the anti--tubulin antibody to reveal DNA (green) and microtubules (red), respectively. Gie-depleted S2 cells displayed no separation of their chromatids before cytokinesis and remained with a thin chromatin string (arrowhead). They also had lagging chromosomes (arrows). Scale bar, 5 µm. (D, left) Representative immuno-stains of control (top) and dGie-dsRNA-treated S2 cells (bottom). The appearance of cells that had a chromatin string (arrowhead) or lagging chromosomes (arrows) at anaphase stage was measured. (D, right) The data are represented as percentages of means±s.e.m. from at least three independent experiments (each of 100 cells).