Flow cytometry and cell cycle analysis

DU145 and PC3 cells (3×10⁵ cells/ml) were transfected/treated with si-CCAT2, si-NC or si-CCAT2 + 20 mmol/l lithium chloride (LiCl; Sigma-Aldrich Merck KGaA) at 37°C for 24 h. After 48 h, cells were centrifuged at 1,000 × g for 5 min at room temperature, washed with PBS and fixed with pre-cooled 70% ethanol at 4°C overnight. Subsequently, cells were stained at room temperature for 30 min with propidium iodide (PI; Sigma-Aldrich; Merck KGaA) staining buffer containing 500 µg/ml PI and 100 µg/ml RNase A at 37°C for 30 min in the dark. Cell cycle distribution was measured using a FACSCalibur flow cytometer (BD Biosciences), and cell cycle profiles were generated using ModFit software (v3.0; BD Biosciences). Cell cycle distribution is presented as the percentage of cells in the G₀/G₁, S and G₂/M phases. Flow cytometry was performed in triplicate.