Figure 2. (A–C) High-content analysis of mitotic arrest.

HeLa cells were treated with vehicle (dashed line) or ten two-fold dilutions of paclitaxel (□), vincristine (■), (−)-pironetin (●), 3 (○), or 4 (▲) for 21 h and analyzed by high-content analysis for (A) microtubule mass, (B) condensed nuclei, and (C) mitotic index. All agents with the exception of 4 caused arrested cells in mitosis. The microtubule-destabilizing agents vincristine, (−)-pironetin, and 3 showed initial increases in tubulin immunostaining that reversed at higher concentrations. In contrast, tubulin immunostaining increased steadily with the microtubule-stabilizing agent, paclitaxel. Synthetic (−)-pironetin was as potent as vincristine and paclitaxel, whereas 3 was about 20-fold less active. Data are the averages from quadruplicate wells from a single experiment that has been repeated twice with similar results. (D–G) Microtubule morphology. Photomicrographs of HeLa cells treated with (D) vehicle (DMSO), (E) vincristine (200 nM), (F) (−)-pironetin (100 nM), or (G) 3 (1.5 μM), and stained for α-tubulin (green), phospho-histone H3 (red), and nuclei (blue). Vehicle-treated cells have highly organized microtubules and a low percentage of mitotic cells. All agents caused a heterogeneous response of microtubule disorganization and possibly loss of microtubule mass, increased numbers of phospho-histone H3 positive cells, as well as chromatin condensation and nuclear fragmentation. Images shown are representative fields from a single experiment that has been repeated twice with similar results. (H and I). Loss of tubulin immunostaining in cells treated with microtubule destabilizing agents occurs in both mitotic and non-mitotic cell populations. Three HCS parameters of cellular activity in 1,000 individual cells were graphically represented using Spotfire Decision Site. (H) Vehicle-treated cells (grey) show baseline tubulin staining (x-axis), a low percentage of mitotic cells (y-axis), and 2N DNA content (z-axis). High concentrations of paclitaxel (100 nM, red) caused mitotic arrest and increased microtubule staining due to formation of bright, stable microtubule bundles. In contrast, mitotic and non-mitotic cell populations in vincristine-treated cells (yellow) were shifted to lower tubulin staining intensity compared with paclitaxel, although the loss was more pronounced in the mitotic cells. (I) Pironetin (100 nM, green) and 3 (2.5 μM, black) show mitotic arrest with reduced microtubule staining in the mitotic cells, similar to vincristine (yellow) and different from paclitaxel (red). All axes are logarithmic scale.