Figure 8

Centrosomes assembly, bifocal spindles and chromosomes segregation were not impaired by PJ-34 in normal human proliferating cells. Epithelial cells MCF-10, mesenchymal stroma cells and endothelial cells (Huvec) were scanned by confocal microscope after 72 hours incubation in the absence or presence of 20 μM PJ-34, applied 24 hours after seeding at the indicated concentrations. Top: Percentage of multi-focal spindles (approximately zero) in randomly selected fixed cells was calculated out of at least 20 detected spindles in 3 different experiments performed with each cell type (MCF-10, black line, solid black square; mesenchymal cells, grey line, solid grey diamond; Huvec, grey line, grey triangle). Control (untreated) and treated cells were permeabilized and immunolabeled for α- and γ-tubulin for the detection of spindles and centrosomes, respectively, as described below. Bottom: Confocal images of randomly selected cells in mitosis, untreated (control) cells, and cells that were incubated with PJ-34 for 72 hours at the indicated concentrations. Untreated cells and cells treated with PJ-34 were permeabilized and immunolabeled for α- and γ-tubulin (green and red, respectively) for detection of spindles and centrosomes, respectively. Chromosomes were labeled with DAPI reagent (blue).