ISWI is required for cell growth, anaphase MT stability, and chromosome segregation. (A) ISWI is down-regulated by dsRNAs in Drosophila S2 cells. Cells were treated with described dsRNAs for 4 d. The cells (each 5 × 104 cells) were loaded on a gel and immunoblotted using anti–N-terminal Xenopus ISWI antibody. *, nonspecific band and loading control. (B) ISWI-depleted cells stop growing. After transfection with described dsRNAs (d 0), the number of the cells was counted using a hemocytometer at the day indicated. At 6 d, the confluent cells were diluted to 0.33 × 104 cells/ml and further incubated with fresh dsRNAs. Error bars represent the SD of two independent experiments. (C) ISWI-depleted cells accumulate in G2/M phases. S2 cells were treated with the described dsRNAs for 12 d, stained with propidium iodide, and analyzed by FACS. (D) Mitotic cells detected in control RNAi cells. At 4 d after dsRNA treatment for GFP, the cells were fixed and stained for tubulin (red), DNA (blue) and phopho-histone H3 (green). Mitotic cells, identified by the mitotic specific phospho-histone H3 signal, were analyzed using a confocal microscope. The images projected with maximum intensity are presented. (E) Abnormal mitotic cells specifically found in ISWI-depleted cells. At 4 d after dsRNA treatment for ISWI (MRC015_H12), the cells were fixed, stained, and analyzed using a confocal microscope. The abnormal spindles were classified into four phenotypes. (1) Longer spindles with chromosomes staying in the center region. (2) Longer spindles with chromosomes mainly staying in the center but some locating close to poles. (3) Longer defective spindles or shrinking spindles having chromosomes scattered through the spindle. (4) Anaphase or telophase spindles judged by chromosome separation with defective MTs and chromosomes lagging or missegregating. In phenotype 3 and 4, MTs were often displaced from spindles by accompanying chromosomes, but were still connected to spindle poles (arrows). Bars, 5 µm.
