Accuri 6 cytometer

Lipid peroxidation levels were measured as previously described^{69 (link)}. Briefly, cells were seeded in triplicate in 12-well plates 1 day before treatment, pretreated with or without drugs for 24 h, and/or then irradiated. After the cells were incubated for 24 or 48 h, the cell culture medium of each well was replaced with a fresh medium containing 5 μM BODIPY 581/591 C11 dye (Invitrogen, D3861) for lipid peroxidation measurements and incubated for 30 min in a humidified incubator (at 37 °C, 5% CO₂). Subsequently, cells were washed with PBS and trypsinized to obtain a cell suspension. Lipid peroxidation levels were analyzed by flow cytometry using an Accuri 6 cytometer (BD Bioscience). The gating strategy used for the assay was shown in Supplementary Fig. 9.

Cell death or cell viability was measured as previously described^{64 (link),65 (link)}. Briefly, cells were seeded into 12-well plates or 96-well plates 24 h before treatment. Cell death after relevant treatments was measured by PI staining followed by flow cytometry analysis. Trypsinized cells were incubated in 100 μl of PBS containing 2 μg/ml of PI for 30 min at room temperature and analyzed with an Accuri 6 cytometer (BD Bioscience). Cell viability was measured using the CCK8 kit (Doijindo, Japan). Cells were seeded into 96-well plates. Following appropriate treatment, cell culture media was replaced with media containing 10% CCK8 reagent. The plate was incubated for 1 h at 37 °C in the CO₂ incubator and the OD value at 450 nm was measured by a microplate reader (FLUOstar Omega, BMG Labtech) to quantify cell viability.

The cells were seeded in six-well plates at 50%–60% confluence and treated as indicated 24 h after plating. After 48 h, the cells were harvested and fixed in 70% ice-cold ethanol overnight and then stained with propidium iodide in the presence of RNase A (Beyotime). Fluorescence intensity was measured with an Accuri 6 cytometer (BD Biosciences). The percentages of cells in G0/G1, S, and G2/M phases were analyzed in ModFit LT software (Verity Software House, Topsham, ME, USA).

The lipid peroxidation of treated cells were measured as previously reported 9 (link). Briefly, the cells were stained with 10uM BODIPY 581/591 C11 dye (Invitrogen, D3861) before flow cytometry in an Accuri 6 cytometer (BD, Bioscience).