Fig. 4

Oxidative stress is critical for NFS1 deficiency-induced PANoptosis under oxaliplatin treatment. a Gene Ontology (GO) enrichment analysis of genes that exhibited twofold upregulation under oxaliplatin treatment (40 μM, 24 h) in the NFS1-knockdown group compared with the control group. b ROS analysis of control and NFS1-knockdown HCT116 and DLD1 cells under PBS or oxaliplatin treatment (40 μM/80 μM, 24 h). c Cell viability of HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC or 5 mM GSH for 24 h after NFS1 knockdown. d Lipid ROS analysis of HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC for 24 h after NFS1 knockdown. e Representative images showing YP1⁺ cells (green) which may undergo apoptosis or necroptosis and PI⁺ cells (red) which may undergo apoptosis, necroptosis, pyroptosis, or ferroptosis among control and NFS1-knockdown HCT116 cells treated with 40 μM oxaliplatin combined with 5 mM NAC (24 h) after NFS1 knockdown. The bottom panel shows representative bright fields and the red arrowheads indicate the large bubbles emerging from the plasma membrane. Scale bar = 100 μm. f ROS analysis of control and NFS1-knockdown HCT116 cells under cisplatin (40 μM, 24 h) or H₂O₂ (100 μM, 24 h) treatment. g, h Cell viability of HCT116 cells treated with 100 μM H₂O₂ (g) and 40 μM cisplatin (h) combined with 5 mM NAC for 24 h after NFS1 knockdown. i Cell cytotoxicity assessments of control and NFS1-knockdown HCT116 cells under cisplatin (40 μM, 24 h) or H₂O₂ (100 μM, 24 h) treatment. j Western blotting analysis of caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, phosphorylated MLKL, total MLKL, GSDME, cleaved GSDME and TFRC expression in control and NFS1-knockdown HCT116 cells after treatment with 40 μM oxaliplatin combined with 5 mM NAC (24 h). k–m Photograph showing the gross comparison (k), tumor volumes (l), and weights (m) of control and NFS1-knockdown HCT116 CDX tumors in nude mice subjected to i.p. injection of oxaliplatin (7.5 mg/kg) and NAC (1 mg/ml) in the drinking water (n = 5). Vinculin was included as a loading control. The data in (b–d, f–i) are representative of three independent experiments and those in (l, m) are representative of five independent experiments, and all are presented as the mean ± SD. The P values in (b, l) were calculated by two-way ANOVA, and those in (c, d, f–i, m) were calculated by one-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001