Cell proliferation assay and clonogenic survival assay

Cell viability was measured with the use of the CCK-8 assay (Bioss, Beijing, China). A 96-well plate was seeded with 2000 cells/well and cultured at 37 °C with 5% CO₂. The cells were pre-treated with 5 μMFerrostatin-1 (Cat#SML0583, Sigma-Aldrich, Missouri, USA) or DMSO (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 24 h before RT. RT was conducted with a cabinet irradiator (Rad Source Technologies, Georgia, USA) at doses of 6 Gy, and a dose rate of 165 MU/min. Cell viability was determined following the manufacturer’s instructions, and the optical density (OD) value was measured at 450 nm using a microplate reader (SpectraMax; Molecular Devices, California, USA). In addition, the blank background group—wells with only DMEM medium was set up to eliminate the OD value of medium. The calculation formula for the cell proliferation rate is: cell proliferation rate (%) = (OD treatment group−OD Blank)/(OD control group−OD Blank) × 100% [22]. Cell survival following radiation exposure was defined as the cell’s ability to maintain clonogenic capacity and subsequently form colonies. Cells were counted and seeded in six-well plates at 500 cells/well. After 24 h pre-treatment with 5 μM Ferrostatin-1, the cells were exposed to the indicated doses (0–6 Gy) of RT and incubated at 37 °C for 12–14 days. Colonies were stained with crystal violet, manually counted, and those consisting of ≥ 50 cells were scored. All experiments were performed in triplicate.