Fig. 1: EBV infection inhibits ferroptosis in NPC cells.

CNE2 EBV-negative or CNE2 EBV-positive cells were seeded into six-well plates. Cells were cultured for 24 h and then subjected to cystine starvation for 30 h. Cell death was assessed using SYTOX Orange staining (A) or PI-Annexin V double staining followed by flow cytometry analysis (B) (n = 3). C Twenty-four hours after cystine starvation and 2 μM ferrostatin-1 (Fer-1) treatment, lipid reactive oxygen species (ROS) production was determined by C11-BODIPY staining followed by flow cytometry (n = 3). D Cell death of EBV-negative or EBV-positive CNE2 cells after treatment with cystine starvation, RSL3, erastin or DMSO (control) for 30 h with or without the caspase inhibitor z-VAD-FAK (n = 3). E. Cell viability of CNE2 EBV-negative or CNE2 EBV-positive cells was determined after treatment with different concentrations of RSL3 or erastin for 30 h by CCK-8 assay (n = 4). F. Lipid ROS production in CNE2 EBV-negative or -positive cells was determined after treatment with RSL3, erastin or DMSO (control) for 24 h (n = 3). G Representative western blots of 4-hydroxynonenal in EBV-negative and EBV-positive cells. GAPDH was used as a loading control. H Subcutaneous tumors formed by CNE2 EBV-negative or CNE2 EBV-positive cells in nude mice were excised 17 days after inoculation. Tumor growth was assessed by volume changes over time and weight at the endpoint (n = 7). I Representative images of immunohistochemistry staining showing high levels of 4-hydroxynonena in CNE2 EBV-negative xenografts. Data are shown as the mean ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001. B–F, two-tailed unpaired t test. H two-tailed Mann–Whitney test. A and I scale bars: 100 µm.