Proliferation and Apoptosis Assays

Triplicate samples of 5,000 cells were seeded in 96-well plates in medium containing puromycin for 48 h before proliferation was assessed using CellTiter 96 (Promega) at 570 nm absorbance using a Synergy-2 multi-detection plate reader (BioTek). Alternatively, cells were incubated with 10 µM bromodeoxyuridine (BrdU) for 2 h, washed in PBS, and fixed in 70% ethanol. DNA was denatured in 2 N HCl at room temperature for 15 min and resuspended in 0.1 M Na₂B₄O₇, followed by staining with FITC-conjugated BrdU Ab (BD Pharmingen), washing in PBS, additional staining with 10 mg/ml propidium iodide for 30 min, and analysis on a FACScan instrument (Becton-Dickinson). For analysis of apoptosis, we used a FITC Annexin V Apoptosis detection kit I (BD Pharmingen) and analyzed cells on a FACScan instrument (Becton-Dickinson). Phospho-Histone H3 was analyzed as described previously^{34 (link)}, using Histone H3 (Cell Signaling, cat. no. 9715) and Phospho-Histone H3 (Cell Signaling, 3377) Ab.