Anticancer effects of the PLK4 inhibitors CFI-400945 and centrinone in Ewing’s sarcoma cells

Figures

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  Fig. 1. Antineoplastic effects of PLK4i in ES cells. Cells were exposed to CFI-400945 or centrinone for 48 h (PI uptake analysis, cell viability assay) and 72 h (cell viability assay). a Cell death was determined by flow-cytometric analysis of PI uptake. b Cell viability was determined by resazurin assay. Means ± SEM of each three separate measurements are shown

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  Fig. 2. Induction of cell death in ES cells by PLK4i involves apoptosis. Cells were exposed to CFI-400945 or centrinone for 24 h (b, c) or 48 h (a, d). a Δψm loss was determined by flow-cytometric analysis of DiOC6(3) staining. b Caspase 3/7 activity was determined using the fluorogenic substrate Ac-DEVD-AMC; relative caspase 3/7 activities are the ratio of treated cells to untreated cells. c PARP1 cleavage was determined by immunoblotting. d Cell death was determined by flow-cytometric analysis of PI uptake; z-VAD-fmk was applied 1 h before treatment with PLK4i. a, b, d Means ± SEM of each three separate measurements are shown (*P < 0.05, **P < 0.01, ***P < 0.001). c The figure is representative of three independent determinations

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  Fig. 3. PLK4i induce cell cycle effects in ES cells. Cells were exposed to CFI-400945 or centrinone for 48 h; z-VAD-fmk was applied 1 h before treatment with PLK4i. Cell cycle profiles were determined by flow-cytometric analysis of PI-stained ethanol-fixed cells. The figure is representative of each three independent measurements

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  Fig. 4. Time course of PLK4i-induced cell cycle effects in WE-68 cells. Cells were exposed to 30 nM CFI-400945 or 2 µM centrinone for the indicated times; z-VAD-fmk was applied 1 h before treatment with PLK4i. Cell cycle profiles were determined by flow-cytometric analysis of PI-stained ethanol-fixed cells. The figure is representative of three independent measurements

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  Fig. 5. PLK4i induce DNA damage in WE-68 cells. Cells were exposed to CFI-400945 or centrinone for 2 h (a) or 24 h (b). a H2AX phosphorylation and b p53 expression levels were determined by immunoblotting. The figures are representative of each three independent determinations. The loading control in b is the same as in Fig. 2c since PARP1, p53 and vinculin were detected on the same blots