PMC

Mitotic asters induced by paclitaxel or the taccalonolides. The mitotic asters in HeLa cells treated with vehicle, 12 nM paclitaxel, 5 μM taccalonolide A or 20 nM taccalonolide AJ, the minimum concentrations that caused maximum mitotic accumulation, were visualized by indirect immunofluorescence for β-tubulin (top). DNA was visualized by DAPI staining (middle) and merged images are shown with microtubules in green and DNA in blue (bottom).

Effects of paclitaxel or taccalonolide A on mitotic accumulation and aster number. The number of mitotic cells present at each time after the addition of (A) 12 nM paclitaxel or (B) 5 μM taccalonolide A to GFP-β-tubulin expressing HeLa cells was counted. These mitotic cells were classified as containing bipolar spindles (black) or multiple asters (gray). Cells from 60 microscopic fields (20x) from 12 individual wells were analyzed for each condition at each time point.

Resolution of drug induced asters. The resolution of the multiple asters formed in GFP-β-tubulin expressing HeLa cells after the addition of 12 nM paclitaxel or 5 μM taccalonolide A were evaluated 5–8 h after drug addition. (A) A representative microscope field showing mitotic cells that undergo: 1) a decrease in aster number (green circle), 2) increase in aster number (yellow circle) or 3) no change in number of asters (red circle) are indicated. (B) Percentage of mitotic cells that undergo each phenotype or successfully divide during the time period. 100 cells were followed over time for each condition.

Aster formation following the addition of paclitaxel or taccalonolide A. GFP-β-tubulin expressing HeLa cells were treated with 12 nM paclitaxel or 5 μM taccalonolide A. Every cell that entered mitosis within 3.5 h after drug addition was followed until 8 h after drug addition and placed in one of 4 categories. (A) Representative images of the 4 categories of cells entering mitosis: bipolar spindle formation followed by cell division, bipolar spindle formation that persisted without completion of mitosis, bipolar spindle that resolved into multiple asters or formation of multiple asters immediately upon mitotic entry. Each image represents approximately 40 μm. The category of cells entering mitosis at the indicated time points following (B) paclitaxel or (C) taccalonolide A addition are shown. Cells in 60 individual microscopic fields from 12 separate wells were analyzed for each condition at each time point. (D) Effects of NuMA depletion on taccalonolide A-induced asters. Microtubules were visualized by indirect immunofluorescence 4 h after the addition of 5 μM taccalonolide A in control (GADPH siRNA) and NuMA-depleted (NuMA siRNA) HeLa cells (right). DNA was visualized by DAPI staining (left).