FIGURE 5.

Tubastatin A altered the assembly dynamics of interphase microtubules in MCF-7 cells. A, tubastatin A delayed the reassembly of nocodazole-depolymerized interphase and mitotic microtubules. Cells were treated with either 500 nm nocodazole for 4 h (interphase cells) or 300 nm nocodazole for 24 h (mitotic cells). Nocodazole was then removed by repeated washing, and microtubules were allowed to reassemble in the absence (control) or presence of 15 or 30 μm tubastatin A for 90 min. Cells were then fixed and processed for co-immunostaining with antibodies against α-tubulin (green) and acetylated tubulin (red) (interphase cells) or α-tubulin (red) and phosphohistone-H3 (Ser-10) (green) (mitotic cells). PH-H3 (Ser-10), a mitotic marker, was used to indicate the chromosomes. Scale bar, 10 μm. B, tubastatin A delayed the reassembly of cold-depolymerized interphase microtubules. Microtubules were depolymerized by incubating the cells on ice for 45 min. Then cells were incubated with prewarmed medium, without or with 30 μm tubastatin A, at 37 °C in incubator for different time intervals (0, 20, 40, 120, and 180 min) and then fixed and processed for immunostaining with antibodies against α-tubulin (green) and acetylated tubulin (red). Scale bars, 10 μm. C, life history plots of the individual microtubules in EGFP-tubulin-expressing MCF-7 cells treated with vehicle or 15 μm tubastatin A for 24 h. The initial length represents length from an arbitrary fixed point of microtubule to the end of the microtubule.