Mod t lt software

The cells in 10 cm dishes were trypsinized, washed and xed with 70% ethanol at the indicated time points and stored at -20°C for at least 12 hours. Cells were then washed and incubated with PI/RNase staining buffer (BD Pharmingen) for 15 minutes at room temperature. The cells were analyzed on the Accuri C6 ow cytometer. The mod t LT software (Verity Software House, Topsham, ME, USA) was used to calculate tted cell cycle values.

Cell cycle of U2OS cells was examined with propidium iodide (PI) kit (CA1510, Solarbio). Transfected U2OS cells (5 × 10 4 /dish) were cultured in 6 cm dishes. After 72 h culture, cells were xated in 70% ethanol overnight at 4°C, washed by PBS and suspended in 100 µL R RNase A solution. Next, after incubation at 37 °C for 30 min, cells were treated with 400 µL PI for 30 min at 4 °C. Cell populations in in each of the cell cycle phases were quanti ed using FACS Caliber cytometer (BD Biosciences) and the data were analyzed by the Mod t LT software (Verity Software House, USA).
For apoptosis experiment, transfected U2OS cells were harvested and dyed by both Annexin V-FITC and PI as manufacturers' protocol (CA1020, Solarbio). Brie y, harvested U2OS cells were centrifuged at 300× g for 10 min. After discarding the supernatant, the cells were resuspended in 1 ml 1 × binding buffer and the cell concentration was adjusted to 1×10 6 cells/ml. 100 µL of cells (1 × 10 5 ) was added to each labelled tube, incubated with 5 µL Annexin V-FITC for 10 min and then incubated with 5 µL PI for 5 min.