Annexin 5

Cells were treated with cisplatin (IC₅₀), and then apoptotic cells were detected using an Annexin V assay (Elabscience, Wuhan, China) after 48 h.

Cells seeded at a density of 2x10⁵ per mL were treated with different concentration of H₂O₂ or AS1842856 (MCE, HY-100596), or cells at 1.5x10⁵ per mL were untransduced, transduced with NT, FoxO1 shRNAs or FoxO3 shRNAs, for three days. Then, cells were pelleted by centrifuging at 1500 rpm and subsequently washed once with PBS, followed by double staining of PI and phycoerythrin-cyanine 7-conjugated Annexin V (Elabscience, 2GB58JNSER) for 15 min at room temperature. Apoptotic cells were visualized by flow cytometry (Thermo Fisher, Attune NxT), and analyzed by FlowJoV10 (FlowJo, LLC, Ashland, OR). Cells were unstained with both Annexin V and PI indicated live cells; cells were stained with Annexin V but not PI suggested early apoptosis; cells were stained with both Annexin V and PI denoted late apoptosis, and cells were stained with PI but not Annexin V indicated dead cells.

Pyrrole, polyethyleneimine, ferric chloride, polyethylene glycol, dichlorodihydrofluorescein diacetate (DCFDA) (Sigma Aldrich, Taipei City, Taiwan), AF (MedExpress), ferrous chloride, sodium hydroxide, hydrochloric acid, N-hydroxy succinimide (NHS), 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC), fetal bovine serum (FBS), phosphate-buffered saline (PBS), Dulbecco’s minimal essential medium (DMEM), trypan blue, cyanine 5 dye, Hoechst 33342, an antibiotic/antimycotic (Thermo Scientific, Taipei City, Taiwan), Annexin V, propidium iodide (PI; Elabscience, Taipei City, Taiwan), and formaldehyde (Bio Man, New Taipei City, Taiwan) were commercially procured.