Although only slightly delayed degradation of cyclin A, cyclin B1-cdk1AF treatment significantly affected the morphological changes of added nuclei hindering the metaphase-to-anaphase transition as well as subsequent chromosome disjunction (Figs. S2, S3).
It should be noted that the present analyses utilized only annulus cells in the microarray expression studies; we currently are adding nucleus specimens so that future work can explore expression patterns in nucleus cells as well as the annulus.
At each timepoint, cells were transferred to a BD Falcon 96-well black clear bottom imaging plate and live-dead viability dyes (calcein - live cells; ethidium homodimer - dead cells) and hoescht 33342 for total nuclei (Invitrogen®) were added in complete medium.
A secondary PE-conjugated antibody (Becton Dickinson) was added and nuclei were counterstained with 1 mM Hoescht 33342 (Fluka).
After passing 10 times through a 40 gauge needle, 400 µL mitochondrial buffer (210 mM mannitol, 70 mM sucrose, 20 mM Hepes-KOH, pH 7.5, and 1 mM EDTA containing 0.45% bovine serum albumin (BSA)) was added and nuclei were pelleted by centrifugation for 1000 xg for 10 min at +4°C.
Then, detergent was added and nuclei were collected by centrifugation (14,000 rpm, 4°C, 30 s).
Crosslinked tissue was washed by 1X PBS and cell lysis buffer with protease inhibitors (PIs) was added and nuclei were prepared by using a dounce homogenizer.
Frozen, ground tissue was added to Nuclei Isolation Fix Buffer (NIFB: 20 mM TRIS-HCl pH 7.8; 250 mM Sucrose; 5 mM MgCl2; 5 mM KCl; 40% Glycerol, 0.1% β-mercaptoethanol [βME]; 0.1 mM PMSF and 1% Formaldehyde).
An equal volume of IP-850+ (10 mМ Tris HCl pH 8.0, 850 mМ NaCl, 10 mМ MgCl2, 1 mМ EDTA, 1 mМ EGTA, 1 mМ DTT, 10%% glycerol, 0.1 % NP-40) was added and nuclei were lysed for 10 min on ice, after that two volumes of IP-10+ and 1 U/ml DNAse I were added and incubation was continued for 10 min in a rotator at room temperature.
Then they added the nucleus from a Cumulus cell, a kind of cell that surrounds the developing eggs in an ovary.
