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. Author manuscript; available in PMC: 2011 Jan 1.
Published in final edited form as: Int J Cancer. 2010 Jan 1;126(1):256–265. doi: 10.1002/ijc.24765

Figure 2.

Figure 2

A. EM011 induces spindle abnormalities in melanoma cells. Panels show immunofluorescence confocal micrographs of B16LS9 cells treated with DMSO vehicle (top row) and 25 μM EM011 (bottom row) for 0, 12, 24, 48 and 72h. As expected, vehicle-treated cells showed normal radial arrays of microtubules (time 0). Their cell-cycles progressed normally over time with bipolar spindle formation and complete chromosome congression at the metaphase plate (12h). During anaphase, separation of sister chromatids was evident (24 and 48h) followed by normal telophase and cytokinesis into two daughter cells. In contrast, EM011-treated cells showed perturbed spindle and nuclear morphologies. At 12h of drug treatment, numerous mitotic figures with multiple asters were evident. Cells with multiple asters either succumbed to apoptosis directly (as evident by the appearance of fragmented nuclei) or exited mitoses without cytokinesis as multinucleate interphase cells and continued to traverse the cell-cycle and accumulated more and more DNA that finally triggered apoptosis due to genotoxicity. B. EM011 inhibits cell-cycle progression at mitosis followed by appearance of a characteristic hypodiploid (sub-G1) DNA peak, indicative of apoptosis. Panels (left and middle) depict cell-cycle distribution in a three-dimensional disposition as determined by flow-cytometry of B16LS9 cells treated with 10 and 25 μM EM011 for 0, 24, 48 and 72h. Right panel is a graphical representation of the quantitation of mitotic index (percent G2/M cells) and apoptotic index (percent sub-G1 cells) at the two dose regimes (10 and 25 μM) over the time course of treatment. Values and error bars shown in the graph represent means and standard deviations, respectively, of three independent experiments performed in triplicate. C. Left panel shows representative Alexa-fluor 488-annexin V stained B16LS9 cells upon 25 μM EM011-treatment showing green rims (solid arrowheads) due to phosphatidylserine staining on the outer plasma membrane in early apoptotic cells as opposed to control vehicle-treated cells, where clear green rings are absent. However, some non-specific staining is evident. Right panel shows quantitation of annexin-positive cells upon EM011 treatment for the noted hours as revealed by flow-cytometric analysis. (P<0.05).