*4.4. Flow Cytometric Analysis*

Flow cytometry was used to analyze cell cycle distribution. Cells were seeded in 60 mm dishes. After 24 h, cells were cultured for an additional 24 h in the absence (control) or presence of JI017 (50–150 μg/mL). Trypsinized cells were washed with PBS and fixed in 95% ethanol containing 0.5% Tween-20 overnight at −20 ◦C. After washing with PBS, the cells were then incubated with 1 U/mL RNase A and 10 μg/mL PI for 30 min at room temperature in the dark. The DNA content in each cell nucleus was determined by a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA), and the cell cycle was analyzed using ModFit LT V2.0 software.

#### *4.5. Annexin V-FITC Apoptosis Assay*

Flow cytometry was used to analyze cell apoptosis. Cells were cultured in 60 mm dishes. After 24 h, cells were cultured for an additional 24 h in the absence (control) or presence of JI017 (50–150 μg/mL). Annexin V-FITC/PI double staining Apoptosis Detection Kit was purchased from Invitrogen (Waltham, MA, USA), and apoptosis assay was performed using a flow cytometer according to the manufacturer's instructions.
