Figure 4

FRAX1036, docetaxel (DTX) and their combination affects microtubule organization, mitosis and cell fate. (A) Spinning-disk confocal images of live U2OS cells expressing red fluorescent protein (RFP)-Tubulin (red) and green fluorescent protein-Histone H2B (green) bottom panel. The top panel is RFP-Tubulin channel alone with an individual cell outline by a dotted line for each condition. Arrows highlight changes in microtubule organization that are characteristic of each treatment. A micronucleated cell is indicated by +. Cells were treated with DMSO, 2.5 μM FRAX1036, 0.2 μM docetaxel, or a combination of 2.5 μM FRAX1036 and 0.2 μM docetaxel for 20 hours before imaging. Scale bar = 20 μm. (B) Duration of mitosis/mitotic arrest of cells treated with DMSO, 2.5 μM FRAX1036, 0.2 μM docetaxel, or a combination of 2.5 μM FRAX1036 and 0.2 μM docetaxel. Cells were followed from time of entering mitosis to the time of division, slippage or apoptosis. Each grey symbol represents a single cell and black bars represent the average. N = 42 mitotic cells imaged from five fields of view. Data is from one of two experiments with similar results. One-way analysis of variance with multiple comparisons showed that all averages are significantly different except for DMSO:FRAX1036. A t-test was performed on FRAX1036 + DTX combination vs DTX alone (P = 0.0002). (C) Distribution of major cell fates after entry into mitosis of U2OS cells treated with FRAX1036, docetaxel and their combination. N = 42 mitotic cells for each treatment condition.