Fig. 1: KEAP1 regulates ferroptosis in a SLC7A11/GPX4-independent manner in lung cancer cells.

a Schematic overview of ferroptosis pathways and ferroptosis inducers (FINs) was used in this study. Ferroptosis is induced by lipid peroxide buildup. SLC7A11 imports extracellular cystine. Intracellular cystine then undergoes reduction to generate cysteine, which is the rate-limiting precursor required for glutathione (GSH) synthesis. GSH serves as a cofactor for GPX4 to detoxify lipid peroxides. Ubiquinol (CoQH₂) can detoxify lipid peroxyl radicals and can be generated from ubiquinone (CoQ) by FSP1 and DHODH on the plasma membrane and inner mitochondrial membrane, respectively. FINs are classified into various classes depending on their targets of action. Class I FIN used in this study, erastin, targets SLC7A11-mediated cystine import. Class II FINs used in this study, RSL3, and ML162, target GPX4 activity. Class III FIN used in this study, FIN56, depletes both GPX4 protein, and CoQH₂. b Protein levels of KEAP1, NRF2, SLC7A11, and GPX4 in H1299 KEAP1 KO cells were determined by western blotting. KEAP1 KO cells were generated by KEAP1 single guide RNA (sgRNA) infection. Vinculin was used as a loading control. sg sgRNA, C control. c–f Cell death upon erastin (c), RSL3 (d), and FIN56 (e) treatment in H1299 KEAP1 KO cells was analyzed by PI staining and cell viability for FIN56 was estimated by CCK8 (f). Ctrl control. g, h Lipid peroxidation levels were determined for H1299 cells treated with RSL3 (g) and FIN56 (h). i, Protein levels of KEAP1, NRF2, SLC7A11 in H1299 SLC7A11 KO, KEAP1 KO, and SLC7A11-KEAP1 DKO cells. j, k Cell death was quantified by PI staining for H1299 SLC7A11 KO (SLC sg), KEAP1 KO, and SLC7A11-KEAP1 DKO (KEAP1 sg + SLC sg) cells by RSL3 (j) and ML162 (k). l, m Lipid peroxidation levels were determined for H1299 SLC7A11 KO, KEAP1 KO, and SLC7A11-KEAP1 DKO cells treated with RSL3 (l) and ML162 (m). n Protein levels of KEAP1, NRF2, GPX4 in H1299 GPX4 KO, KEAP1 KO, and GPX4-KEAP1 DKO cells. o–q Cell death was quantified by PI staining upon ferrostatin-1 withdrawal for H1299 cells (o) and lipid peroxidation levels were determined (p, q). Data were presented as (if mentioned otherwise) mean ± SD; n = 3. P value was determined by two-way ANOVA; ns not significant. Source data are provided as a Source Data file.