Cells were trypsinized and then terminated by adding equal volume of medium, followed by centrifugation at 4 °C for 2 min (200 × g). Cell pellet was washed twice with cold PBS and fixed in 70% ethanol at 4 °C overnight. Ethanol was removed and cell pellet was washed twice with cold PBS. Cells were then stained with PI/Triton X-100 staining solution (0.1% (v/v) Triton X-100, 0.2 mg/mL DNase-free RNase A (Sigma), 0.02 mg/mL propidium iodide (PI, Roche)) at 37 °C for 15 min. DNA content was then measured by Attune NxT Flow Cytometer (Invitrogen) and about 105 events were analyzed for each sample. Data were analyzed using ModFit LT (Verity Software House).

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