Flow cytometry analysis of the cell cycle

Cells (2×10⁵) were cultured in 6-well dishes. Following attachment, cells were treated with JQ1 (10, 20, 40 µM for DU145, 100, 200, 400 nM for LNCAP) for 48 h or transfected with shBRD4 as aforementioned. Cell suspensions were harvested using 0.25% trypsin for 45 sec at 37°C and placed in precooled 70% ethanol for fixation at −20°C overnight. Then, a ribozyme (50 µg/ml) and propidium iodide (50 µg/ml, PI; BD Biosciences, San Jose, CA, USA) were added to the cells, which were incubated in the dark at room temperature for 30 min. Samples were subsequently evaluated with a flow cytometer (BD FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed by ModFit LT 2.0 software (Verity Software House, Inc., Topsham, ME, USA).