Conventional cytogenetics techniques were performed as described in [7 (link)] in order to determine the effect of 48 h of treatment with 500 nM Danusertib on the mitotic index and cell ploidy. Briefly, cells were treated with 0,2 μg/ml of Colcemid (Roche) and then harvested and incubated with a hypotonic solution of 0,56% w/v of KCl for 15 minutes at room temperature. Subsequently, cells were fixed with a fixative solution composed of 3:1 methanol:acetic acid. Chromosomes were QFQ banded using quinacrine mustard (Roche) and slides were mounted in McIlvaine buffer. Slides were analysed using Nikon Eclipse 80i fluorescence microscope (Nikon) equipped with a COHU High Performance CCD camera. The mitotic index was evaluated counting the percentage of mitosis scoring at least 1000 nuclei, while ploidy was investigated by evaluating the number of chromosomes/metaphase on 30/50 metaphases. Chromosomes spreads were analysed using the Genikon software. Data were obtained as mean values derived from two independent experiments.
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