Cell apoptosis assay kit

For cell apoptosis assay, a cell apoptosis assay kit (Beyotime Biotechnology, China) based on Annexin V-FITC and propidium iodide (PI) dual staining was used on a FACS flow cytometry (Celesta^TM; BD, San Jose, CA, USA). The cell populations were discriminated according to their position of quadrants as we previously reported [14 (link)]: live cells (Annexin V−/PI−), early/primary apoptotic cells (Annexin V+/PI−), late/secondary apoptotic cells (Annexin V+/PI+), and necrotic cells (Annexin V−/PI+).

Cell apoptosis was performed with the cell apoptosis assay kit (Beyotime). Briefly, NCI-H460 cells (5 × 10⁵/per well) were plated in 6-well plates and cultured under hypoxic conditions (37 °C, 5% CO₂/1% O₂/94% N₂) for 24 h prior to RT. The cells in RT+Gly-PFOBs (O₂) 0 h, RT+Gly-PFOBs (O₂) 1~2 h, RT+Gly-PFOBs (O₂) 3 h and RT groups were subjected to RT at the dosage of 6 Gy. The control group was not subjected to any intervention. Cells were collected the next day, washed twice with cold PBS, and centrifuged at 300 × g for 5 min at 4 °C. Subsequently, cells were resuspended in 195 μL of Annexin V -FITC binding buffer and incubated with 5 μL of Annexin V-FITC and 10 μL of PI staining solution at room temperature for 10–20 min. The stained cells were examined using a flow cytometer (Beckman Coulter, Fullerton, CA, United States). The results were analyzed with FlowJo 10 software (FlowJo, LLC). A figure exemplifying the gating strategy is provided in the Supplementary Information (Supplementary Fig. 12c). The MTT assay was used to measure the viability of hypoxic NCI-H460 cells after being subjected to RT at different doses (0 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy) with the addition of oxygenated Gly-PFOBs (O₂).