Isis software

Chromosome preparations were obtained from logarithmically growing HCT116 cell cultures after been exposed to Colcemid (0.1 µg/ml) (Gibco, Grand Island, USA) for 1–3 h, at 37 °C in 5% CO₂. Cells were harvested by trypsinization (Gibco, Grand Island, USA), exposed in hypotonic solution (5 ml of 0.075 M KCl, Sigma), and fixed [3× methanol (Applichem GmbH, Darmstadt, Germany)/1× CH3COOH (Merck, Darmstadt, Germany)]. For karyotypic analysis^{61 (link)}, we applied inverted DAPI banding: slides were stained and mounted with 0.6 μg/ml DAPI in Vectashield antifade medium (Vector Laboratories, USA). Cytogenetic analyses were performed using a 63× magnification lens on a fluorescent Axio-Imager Z1, Zeiss microscope, equipped with a MetaSystems charge-coupled device (CCD) camera, and the MetaSystems Isis software (MetaSystems GmbH, Germany). Karyotypes were recorded according to International System for Human Cytogenetic Nomenclature 2016. Micronuclei analysis was performed in cytologic preparations treated as above and stained by DAPI.

Actively growing hiPSC colonies (80% confluency) were treated with colchicin (20 mg/ml; Eurobio) for 90 min at 37 °C. Cells were dissociated with 0.05% trypsin-EDTA and incubated in 75 mM KCl (Sigma-Aldrich) for 10 min at 37 °C, before fixation with 3:1 methyl alcohol/glacial acetic acid. Fixed cells were hybridized overnight at 37 °C with a denatured “cocktail painting mFISH” probe (MetaSystems). Slides were washed in successive baths of 1× SSC and 0.4× SSC, and nuclei were stained with 250 ng/ml 4’,6-diamidino-2-phenylindole (DAPI). Biotinylated probes were revealed using a Cy5 MetaSystems B-tect detection kit (MetaSystems). 10 to 20 metaphases were captured using a Zeiss Z1 fluorescence microscope equipped with a UV HBO 100-W lamp coupled to an AxioCam camera (Carl Zeiss). All of the analyzed metaphases were karyotyped using the MetaSystems Isis software (MetaSystems).

For cytogenetic analysis, MO59K cells were treated with 1.0 μM of MST-312 and/or 10 μM of NU7026 for 1 week and cells were arrested at mitosis by treatment with colcemid (0.1 mg/ml). Cells were subsequently incubated with a hypotonic solution of potassium chloride at 37°C for 15 minutes followed by fixation in Carnoy’s fixative. Fluorescence in situ hybridisation (FISH) was performed using telomere sequence-specific peptide nucleic acid (PNA) probe labelled with Cy3 as described [44 (link), 45 (link)]. Metaphase spreads were captured using Zeiss Axioplan 2 Imaging fluorescence microscope and analysed using ISIS Software (Metasystems, Germany) for telomere-mediated chromosome alterations.

FISH analysis was performed on cytology specimen fixed in methanol or 3.7% formaldehyde prior to hybridization of a dual color probe (Texas Red-labelled EGFR DNA and green fluorescein-labelled CEN-17 PNA) according to the manufacturer's instructions (DAKO). The samples were washed in stringency buffer and stained with DAPI antifade. Axioplan 2 MetaSystems CoolCube1 camera and DAPI-counterstained band images were used for cytogenetic analysis with the Isis software (MetaSystems, Germany).