At least 104-106 cells were analyzed by flow cytometry with the Annexin V/PI kit (BD Biosciences) using a Beckman Coulter MoFlo XDP. Briefly, the medium was removed and cells were treated with serum-reduced medium (0.5% FBS) with GYY4137 (100 μM) or NAC (5 μM) for 24 h. Cells were suspended in 300 μl binding buffer, and then stained with Annexin-FITC and propidium Iodide (PI). Positive controls for cell apoptosis or necrosis were indicated by staining with only Annexin-FITC or PI (Beckman Coulter, Inc.). Cell apoptosis was counted using summit V5.2.0.7477 software.

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