Cell Cycle and Apoptosis Analysis

Cell-cycle distribution by PI staining was performed as previously described17 (link). Briefly, 50,000 cells were cultured in 6-well plates, treated for 72 h with concentrations ranging from 1 to 50 μM, collected by centrifugation, fixed in cold 70% ethanol and stained with a PBS solution containing PI (62.5 mg/ml; Sigma-Aldrich) and RNase A (1.125 mg/ml; Sigma-Aldrich). Samples were acquired with C6 Accuri Flow cytometry (BD Bioscience) and the percentage of cells in the different phases of cell cycle and in the sub-G1 compartment was calculated. Apoptosis was quantified by cytofluorimetric analysis staining cells simultaneously with FITC-annexin V and the nonvital dye PI (Immunological Sciences, Rome, Italy), following the manufacturer’s instruction. At the end of incubation with the respective reagents, samples were analyzed with C6 Accuri Flow cytometry (BD Bioscience). About 20,000 events were acquired and gated using forward scatter and side scatter to exclude cell debris. Bivariate analysis allows the discrimination of viable cells (FITC−/PI−), early apoptotic (FITC+), and late apoptotic or necrotic cells (FITC+/PI+).