Supplemental Figure 1
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Fig. S1. ZM2 phenocopies ZM447439 at lower concentrations. (a) Immunoblots of HeLa extracts following exposure to increasing ZM concentrations showing that in cells, ZM2 inhibits histone H3 (Ser10) phosphorylation 30 times more potently than ZM1. (b) Mitotic cells were isolated after a 12 hour nocodazole block, washed and then re-plated in vehicle only (DMSO), nocodazole, 2 μM ZM1 or 0.2 μM ZM2 for up to 4 hours, and the mitotic index determined by flow cytometry. Line graph shows that like ZM1, ZM2 accelerates mitotic exit. Each value represents the mean ± s.e.m. derived from three independent experiments. (c) Asynchronous cells were exposed to nocodazole or taxol plus ZM compounds (0.001-10 μM) for 16 hours then analysed by flow cytometry to determine the mitotic index. Line graphs show that ZM2 preferentially overrides taxol-induced mitotic arrest at a lower concentration than ZM1. The data is derived from a representative experiment in which at least 10,000 cells were analysed per concentration. (d) Immunofluorescence images of DLD-1 cells exposed to 2 μM ZM1 or 0.2 μM ZM2 for 2 hours showing that like ZM1, ZM2 causes chromosomes to align along the length of the spindle. Bar, 5 μm.
