5 mm diameter coverslips

CHO-K1 cells (Sigma-Aldrich) were grown in Petri dishes containing F12 medium (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (Invitrogen) and penicillin-streptomycin-glutamine (100 U/ml, 100 µg/ml, 292 µg/ml) and maintained in a humidified 5% CO₂/95% air atmosphere. Cells were electroporated (Nucleofector II; Amaxa) with the plasmids mentioned above (4 µg DNA per reaction) using the Nucleofector kit V (Lonza) according to the manufacturer’s instructions and seeded onto 5-mm-diameter coverslips (Warner Instruments). It is important to note that at least 4 µg DNA was required per electroporation to measure a robust Ae4 activity in CHO-K1 cells 18–20 h after electroporation.

After keeping the cells at 100% confluency for 1 d (a condition that arrests the cells at the end of G1; Coupin et al., 1999 (link); Kozik et al., 2010 (link)), they were trypsinized and seeded onto 5-mm-diameter coverslips (Warner Instruments) that were cleaned by sonication in 95% ethanol at ∼30–50% confluency; this procedure helped to synchronize cell division and increase the proportion of cells entering S phase. The cells in prometaphase and metaphase were identified by their spherical appearance 11–16 h after plating. The cells were then imaged in Leibovitz’s L-15 medium without phenol red (21083-027; Life Technologies) supplemented with 5% FBS and 20 mM HEPES, pH 7.4.