Kaluza software version v2

To analyze the underlying mechanism of the aspirin-mediated inhibition of tumor cell growth, flow cytometric analyses were performed using the Cycle Phase Determination kit (Cayman Chemical Co.). HCC Huh-7 cells (1.0×10⁶ cells/100-mm diameter dish) were treated with 2.5 mmol/l aspirin for 24 to 48 h. Cells were scraped and centrifuged to obtain the cell pellet, which was resuspended in phosphate-buffered saline (PBS) (10⁶ cells/ml). An equal volume of cell suspension was added to the cycle phase determination fixative and stored at −20°C until further analysis. For cell cycle analysis, the cells were suspended in 100 µl of PBS with 10 µl RNase A (250 µg/ml) and 10 µl propidium iodide (PI) stain (100 µg/ml), followed by incubation at room temperature in the dark for 30 min. Flow cytometry was performed to compare the proportion of aspirin-treated and control cells in each phase of the cell cycle. Flow cytometry was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter) with an argon laser (488 nm), and the percentages of cells were analyzed using Kaluza software version v2.1 (Beckman Coulter). The experiments were repeated thrice.

Aspirin-mediated apoptosis was analyzed using flow cytometry and the Annexin V-FITC Early Apoptosis Detection kit (Cell Signaling Technology, Inc.). Briefly, HCC Huh-7 cells (1.0×10⁶ cells/100-mm dish) were treated with 2.5 mmol/l aspirin for 48 h at 37°C. Cells undergoing apoptosis and necrosis were analyzed by double staining with FITC-conjugated Annexin V and PI as per the manufacturer's protocol. This staining method is based on the binding of Annexin V to apoptotic cells with exposed phosphatidylserines, and the PI-labeling of the damaged membrane in late apoptotic/necrotic cells. Flow cytometry was conducted using a Cytomics FC 500 flow cytometer (Beckman Coulter) with an argon laser (488 nm) and data were analyzed using Kaluza software version v2.1 (Beckman Coulter). The experiments were repeated thrice to compare the proportion of apoptotic cells in the aspirin-treated and control groups.

Flow cytometric analyses were performed using the Cycle Phase Determination kit (Cayman Chemical Company). HuCCT-1 and TKKK cells (1.0×10⁶ cells/100-mm dish) were treated with 2.5 mmol/l aspirin or without for 24 to 48 h. Cells were trypsinized and resuspended in phosphate-buffered saline (PBS) at a density of 1×10⁶ cells/ml. Approximately 1×10⁶ cells were stained in 100 µl of PBS with 10 µl RNase A (250 µg/ml) and 10 µl propidium iodide (PI) stain (100 µg/ml) and incubated at room temperature in the dark for 30 min. Flow cytometry (FCM) was performed to compare the proportion of aspirin-treated and control cells in each phase of the cell cycle. FCM was performed using a Cytomics FC 500 flow cytometer (Beckman Coulter, Inc.) with an argon laser (488 nm), and the percentages of cells were analyzed using the Kaluza software version v2.1 (Beckman Coulter, Inc.). The experiments were repeated thrice.