TACC3 and ch-TOG protein levels are reduced by siRNA treatment. A Western blot showing the levels of TACC3 (A) and ch-TOG (B) after 48 h of siRNA treatment. Actin is shown as a loading control. (A) Mock-depleted cells (lane 1), ch-TOG-depleted cells (lane 2), TACC3-depleted cells (lane 3), and TACC3- and ch-TOG-depleted cells (lane 4). (B) Mock-depleted cells (lane 1), TACC3-depleted cells (lane 2), ch-TOG-depleted cells (lane 3), and ch-TOG- and TACC3-depleted cells (lane 4).

The localization of ch-TOG is disrupted in TACC3-depleted cells. (A) The distribution of ch-TOG (left panels, red in merged image) and microtubules (middle panels, green in merged image) in mock-depleted (top panels) and TACC3-depleted (bottom panels) cells. Ch-TOG is detectable on centrosomes, but is not detectable on spindle microtubules in the TACC3-depleted cells. (B) The distribution of TACC3 (left panels, red in merged image) and microtubules (middle panels, green in merged image) in mock-depleted (top panels) and ch-TOG-depleted (bottom panels) cells. TACC3 remains concentrated on spindles in the ch-TOG-depleted cells. Bar, 10 μm.

Immunofluorescence analysis of TACC3 and ch-TOG protein levels in siRNA-treated cells. (A) Mock-depleted (top panels) and TACC3-depleted (bottom panels) HeLa cells stained to reveal the distribution of TACC3, DNA, and microtubules. In this and all subsequent experiments, images were acquired from experimental and control cells that were treated and processed for immunofluorescence at the same time using identical settings on the confocal microscope. The TACC3 siRNA treatment has strongly reduced TACC3 levels in two mitotic cells (arrows), but substantial levels of TACC3 remain in one mitotic cell (arrowhead). (B) Mock-depleted (top panels) and ch-TOG-depleted (bottom panels) HeLa cells stained to reveal the distribution of ch-TOG, DNA, and microtubules. The ch-TOG siRNA treatment strongly reduced ch-TOG levels in most cells visible in this field, but small amounts of protein are still detectable on the centrosomes and spindles of some mitotic cells (arrowhead). Bar, 10 μm.

The depletion of TACC3 and ch-TOG partially destabilizes spindle microtubules. (A) A graph showing the relative spindle fluorescence intensity in nondepleted or TACC3-depleted mitotic cells (black bars), or in nondepleted or TACC3-depleted cells that have been chilled and allowed to recover for 25 min (hatched bars). Note that the spindle fluorescence of both sets of nondepleted cells has been normalized to 1. (B) A graph showing the percentage of cells that have properly aligned the majority of their chromosomes at a metaphase plate after 25 min of recovery from cold treatment. Error bars represent the standard deviation. (C, top panels) A field of cells treated with TACC3 siRNA. One mitotic cell (marked with *) has not been significantly depleted of TACC3, but two other mitotic cells (marked with arrowheads) have been significantly depleted. Fields such as these were used to compare the spindle microtubule density in the depleted and nondepleted cells (see Materials and Methods). Fields of TACC3 (middle panels) and ch-TOG (bottom panels) siRNA-treated cells that were chilled to depolymerize the microtubules and then allowed to recover for 25 min. In each panel, a nondepleted mitotic cell is marked with *, and depleted cells are marked with arrowheads. Bar, 10 μm.

Mitotic defects in TACC3- and ch-TOG-depleted HeLa cells. (A,B) Mock-depleted (left panels) and either TACC3-depleted (A, right panels) or ch-TOG-depleted (B, right panels) cells were stained to reveal the distribution of microtubules (green in merged image), TACC3 or ch-TOG (blue in merged image), and DNA (red in merged image). Bar, 10 μm. (C) Quantitation of the defects observed in TACC3- and ch-TOG-depleted cells. In all graphs, the percentage of cells exhibiting a particular phenotype is indicated for mock-depleted (blue bars), TACC3-depleted (red bars), ch-TOG-depleted (yellow bars), or TACC3- and ch-TOG-depleted (orange bars) cells. (Graph 1) The mitotic index in the population of cells as determined by phospho-histone H3 staining. (Graph 2) The percentage of interphase cells exhibiting multiple or satellite nuclei. (Graph 3) The percentage of mitotic cells in a prometaphase-like state (with no discernible metaphase plate). (Graph 4) The percentage of mitotic cells with a normal metaphase plate. (Graph 5) The percentage of mitotic cells with a clear metaphase plate, but with at least one chromosome not aligned at the equator. (Graph 6) The percentage of mitotic cells with more than two spindle poles. Two-hundred-fifty cells were scored from each of four independent experiments for both the mitotic index and the multinucleated phenotype. Fifty metaphase cells from two independent experiments were scored for the prometaphase, metaphase, and lagging chromosome phenotypes. Thirty mitotic cells were scored from each of four independent experiments for the presence of multipolar spindles. Error bars represent the standard deviation.