FIGURE 4.

Fatostatin induces spindle assembly checkpoint activation and mitotic catastrophe. A–D, immunofluorescence microscopy of HeLa cells treated with DMSO or Fatostatin for 20 h and stained for DNA, tubulin, and AurB (A), INCENP (B), survivin (C), or BubR1 (D). Scale bar = 5 μm. E, HCT116-GFP-H2B cells were arrested in G₁/S and released into media with DMSO, 10 μm PF-429242, 10 μm Betulin, 2.11 μm Fatostatin, or 100 nm Taxol. Cells were imaged every 10 min using time-lapse microscopy, and snapshots of representative cell divisions are shown. Time is in minutes. Thy, thymidine. F, quantification of the percentage of cells undergoing normal or defective (multipolar) cell divisions or cell death (during mitosis or after a failed mitosis) that were treated with the drugs indicated in E. PF, PF-429242; Fato, Fatostatin; Bet, Betulin. Data represent the average ± S.D. of three independent experiments, 40 cells counted for each. G, HeLa cells were treated with DMSO, 2.11 μm Fatostatin, 10 μm PF-429242, or 100 nm Taxol for 24 h, and caspase 3/7 activity was monitored. Data represent the average caspase 3/7 activity in RLU ± S.D. of three independent experiments. H, same as in E, except that 2.11 μm Fatostatin or 100 nm Taxol was added 6 h after G₁/S release, just prior to mitotic entry, and cells were imaged every 5 min. I, same as in E, except that cells were arrested in mitosis with 2.11 μm Fatostatin for 16 h and released into fresh medium before imaging. See also supplemental Movies S1–S8.