2.2. Cell proliferation, cell cycle, and apoptosis assays

Cell proliferation following treatment with the BET bromodomain inhibitor, JQ1 (Sigma, St. Louis, MO), was assessed using the Cell Counting kit-8 (Dojindo, Japan) according to the manufacturer’s recommendation. 15,000 cells/well were plated in a 96-well plate with 1 μM of JQ1 or vehicle DMSO (Sigma, St. Louis, MO) in 100 μl of growth media. Cell viability was measured by addition of Cell Counting kit-8 reagent and luminescence measurement on a Tecan infinite M200 plate reader (Tecan, Switzerland).

Cell cycle analysis was performed 72 h after JQ1 treatment using BrdU flow kit (BD Pharmingen, San Diego, CA) according to the manufacture’s instructions. The cells were resuspended in ice cold 70% ethanol and fixed for 30 min. The cells were washed twice with PBS, then incubated for 30 min at room temperature with 0.5% RNase A and stained with 25 μg/ml propidium iodide (PI; BioLegend, San Diego, CA). Cell cycle data was obtained by using LSRII (BD Biosciences, San Diego, CA).

Apoptosis was analyzed with Annexin V and PI. Cells were incubated with Annexin V binding Buffer (BD Pharmingen, San Diego, CA), 2 μl of Annexin V-APC (BD Pharmingen, San Diego, CA) and 5 μg/ml PI. After 15 min incubation at room temperature, the cells were analyzed.