Modfit lt 2

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).

A cell cycle analysis was performed with propidium iodide (PI; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) staining, as described previously (26 (link),27 (link)). The cells were collected and fixed in 70% (v/v) ethanol (cat. no. 5054.2; Carl Roth GmbH & Co., KG, Karlsruhe, Germany) for 15 min at room temperature, then washed with PBS and suspended in a PI staining solution (50 mg/l; Biolegend, Inc., San Diego, CA, USA; cat. no. 421301) supplemented with 0.1% Triton X-100 and Ribonuclease A (0.25 mg/ml; Sigma-Aldrich; Merck KGaA). The cells were incubated at 37°C for 30 min and then the cell fluorescence was measured with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). The cell cycle analysis was performed with ModFit LT 2.0 software (Verity SoftwareHouse, Topsham, ME, USA).
The cell apoptosis assay was performed with an Annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis detection kit (BestBio, Co., Shanghai, China) as described previously (28 (link)). The treated hOMF cells (1×10⁶ cells/ml) were re-suspended with a1X binding buffer (100 µl), and then double-stained with 20 µg/ml Annexin V-FITC and 50 µg/ml PI for 15 min at room temperature. Cell apoptosis levels were then detected by flow cytometry with version 5.1 Cell Quest Pro software (BD FACSCalibur, San Jose, CA, USA).

The cell cycle distribution of cells with different treatments was determined by flow cytometry. Osteoblast cells were grouped as described above and then inoculated in a 96-well plate at 1.0 × 10⁵ cells/well in 200 μL of culture medium. Following incubation with drugs, the cells were dissociated with 0.25% trypsin without EDTA (Gibco, USA), and FBS was used to stop trypsin activity. After being centrifuged at 1000 g for 4 min, the supernatant was discarded. The cells were resuspended in PBS solution, followed by 1 h of fixation with ice-cold 70% ethanol, washing with PBS again, and 30 min of incubation (at 37°C) with 1 mg/mL RNase A (Sigma-Aldrich, USA). After that, they experienced suspension in 0.5 mL of propidium iodide (PI)/RNase Staining Buffer, followed by 15 min incubation at room temperature. Afterward, a FACS Calibur cytometer (Becton Dickinson, Mountain View, CA) was adopted to analyze stained cells. CELLQuest 3.3 software (Becton Dickinson, Mountain View, CA) was employed to acquire samples at a low flow rate, and ModFit LT 2.0 software (Verity Software House, Topsham, ME) was utilized for analyzing list mode data. Three independently repeated experiments were conducted.