Figure 2

De-clustering of supernumerary centrosomes in mitosis preceded PJ-34 induced cell death in human mammary cancer cells MDA-231. (a) PJ-34 induced multi-polar spindles with scattered centrosomes and cell death in MDA-231 cells. Top: (Left) loss of cell viability after treatment with PJ-34 was quantified by luminescent detection of ATP levels in the treated cells (% ATP levels in cells treated with PJ-34 relative to ATP levels of untreated, control cells) (Methods). Cells were incubated with PJ-34 (applied once at 24 hours after seeding) for 72 hours (grey line) and 96 hours (black line). The mean values and standard deviations of 4 measurements in 3 different experiments are presented. (Right) The percentage of distorted spindles with scattered centrosomes in cells treated with PJ-34 was calculated out of 20 detected spindles by confocal scanning of randomly selected MDA-231 cells that were incubated with PJ-34 at the indicated concentrations and durations (48 hours; grey line) and (72 hours; black line). Both untreated and treated cells were permeabilized and immunolabeled for α- and γ-tubulin in 3 different experiments, as explained below. Middle: Confocal images presenting scattered centrosomes and aberrant chromosomes arrangement in multifocal spindles of randomly selected MDA-231 cells that were permeabilized and immunolabeled for α- and γ-tubulin for the detection of spindles and centrosomes (green and red fluorescent labeling, respectively), 18 hours after application of PJ-34 at the indicated concentrations. Chromosomes were labeled with DAPI reagent (blue) (Methods). Bottom: Double immunolabeling of centrosomes for γ-tubulin (green) and centrin1 (red) in randomly selected MDA-231 cells. Bipolar clustering of 4 centrosomes in an untreated cell (Left) and 4 un-clustered centrosomes in a cell treated with PJ-34 (20 μM) (Right) (Methods). (b) Live confocal imaging indicated the effect of PJ-34 on bi-polar centrosomes' clustering in randomly selected live MDA-231 cells. MDA-231 cells were transfected with vectors encoding for histone H2b-RED (red; for chromosomes labeling) and for γ-tubulin-GFP (green; for centrosomes labeling). Randomly selected cells were scanned overnight by confocal microscopy without or during treatment with PJ-34 (Methods). Upper frame- Un-clustered centrosomes (Left) during mitosis preceded cell death (Right) in a randomly selected live MDA-231 cell that was incubated with PJ-34 (20 μM) for 18 hours before scanning and during scanning (15 hours; imaging is included in Additional File 1). Lower frame- MDA-231 cells untreated with PJ-34. (Left) Live confocal images of randomly selected live MDA-231 cell with clustered centrosomes in anaphase. (Right) confocal image of randomly selected untreated fixed MDA-231 cell in anaphase. This cell was permeabilized and immunolabeled for α-tubuline (green; spindles) and for γ-tubuline (red; centrosomes). Chromosomes were labeled with DAPI reagent (blue).