All treatments were carried out using a six-well plate, and all flow cytometry data were acquired using BD FACSAria II, BD FACSCanto II, and BD LSRFortessa (BD Biosciences). A total of 10,000 counts were acquired for each experimental condition, and all flow cytometry data were analyzed with FlowJo data analysis software (version 10.1) (FlowJo LLC, Ashland, USA).

Cell death was quantified by measuring annexin V–fluorescein isothiocyanate (FITC) binding to externalized phosphatidylserine (with a 488-nM laser) and Draq7 uptake in the cell (with a 561-nM laser). Cells were collected and washed in PBS before resuspension in annexin buffer (BD Biosciences) and incubation with annexin V and Draq7 for 20 min before analysis.

For cell cycle analysis, cells were incubated with the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) (20 μM) for 90 min to quantify cells within S phase and FxCycle violet stain (4′,6-diamidino-2-phenylindole, dihydrochloride), which binds to double-stranded DNA, to determine populations of cells within G1 and G2 phases of the cell cycle. Cells were collected and fixed in ice-cold 70% ethanol. EdU incorporation within fixed cells was quantified using Click-iT EdU Flow Cytometry Assay Alexa Fluor 647 azide (Life Technologies), following the manufacturer’s protocol, and detected with a 640-nM laser. To additionally quantify the population of cells within G1 and G2 stages of the cell cycle, FxCycle violet stain (Invitrogen) was incubated with EdU-incorporated cells for 16 hours at 4°C and detected with a 405-nM laser.

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