Metaphase chromosome spreads preparation

To induce mitotic arrest, 2.5 × 10⁵ cells were treated with 0.5 µg/ml demecolcine (Sigma-Aldrich) for 1 hr at 24 degrees. Cells were then pelleted by centrifugation for 5 min at 600 g at room temperature and resuspended in hypotonic solution (250 ml of 0.5% sodium citrate), and incubated for 8 min. Following incubation, cells were placed in a cytofunnel and spun at 1,200 rpm for 5 min with high acceleration using a cytocentrifuge (Shandon Cytospin 4; Thermo Fisher Scientific). Spreads for FISH were immediately fixed in cold 3:1 methanol: acetic acid for 10 min, while spreads for IF were fixed with 4% PFA for 10 min. Following fixation, all slides were washed 3 times for 5 min in PBS-T (PBS with 0.1% Triton X-100).