*2.5. Cell-Cycle Analysis*

Cells (8 <sup>×</sup> 105) were seeded in 6-well dishes. After treatment, cells were trypsinized, washed in PBS, and centrifuged at 2000× *g* at 25 ◦C for 5 min, and then they were washed with PBS at least twice. Cells were fixed in 70% ethanol overnight. Before removing the ethanol, samples were centrifuged at 11 ◦C and 2200× *g* for 10 min. The pellet was then resuspended in 200 μL of DNA extraction buffer (containing 192 mL 0.2 M Na2HPO4 and 8 mL 0.1 M citric acid at pH 7.8) and incubated for 30 min at 37 ◦C. PI dye (200 μL, containing 0.1% Triton-X100, 100 μg/mL RNase-A, and 80 μg/mL PI in PBS) was

added, gently mixed, and incubated for 30 min at room temperature in the dark. After removing the PI dye, samples were resuspended with 1 mL of cold PBS prior to analysis by flow cytometry.
