Actinomycin d

Metaphase prep for cell culture was modified from the preparation of metaphase chromosomes of adherent cells (NCI National Institute of Health, 2006 ). All incubations were for 15 mins and all centrifugations were performed for 8 mins at 350xg. First, 300μl of Actinomycin D (AG Scientific, San Diego, CA) at 100μg/mL was added to the flask and incubated for 15mins. Next, 300μl KaryoMax® Colecmid® Solution (Gibco® Life Technologies, Grand Island, NY) was added to the flask. FISH protocols were adapted from Coriell. The centromeric probe used was CEP 11 (D11Z1) SpectrumGreen probe 11p11.11-q11 Alpha Satellite DNA (06J37-021 Abbott Molecular, Abbot Park, IL). In addition, the slides were stained with ProLong gold antifade reagent with DAPI (P36935 Molecular Probes Life Technologies Grand Island, NY) and stored at −20°C. Images were taken at 63x on a Leica CTR microscope using Perkin Elmer Volocity 6.1.1.

HeLa cells (ATCC, Manassas, VA, Cat. # CCL-2) were maintained in culture at 37°C in a humidified incubator with 5% CO₂ in high glucose Dulbecco's modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) and 1% penicillin-streptomycin (Invitrogen). On the day of the experiment, cells were placed in serum-free medium (DMEM) or leucine-free medium (Atlanta Biologicals, Lawrenceville, GA) for 10 hours. After 10 hours of starvation, 10% serum or 0.76 mM leucine (Sigma, St. Louis, MO) was re-introduced into the culture medium for the indicated times. For experiments using Actinomycin D (Sigma) or cycloheximide (A.G. Scientific, San Diego, CA), cells were prepared as described above with the exception that either Actinomycin D (2ug/mL) or cycloheximide (100ug/mL) was added to the medium for 15 min or 2 min, respectively, prior to the re-introduction of serum or leucine.