Fig 2: Suppression of lenvatinib‐induced ferroptosis by nuclear factor erythroid‐derived 2‐like 2 (Nrf2) in hepatocellular carcinoma (HCC) cells. (A) HCC cells were transfected with siRNA (Nrf2 and control), and Nrf2, phosphorylated Nrf2 (p‐Nrf2), and NADPH quinone oxidoreductase 1 (NQO1) protein levels were assayed via immunoblotting. (B) HCC cells were transfected with siRNA (Nrf2 and control) and treated with lenvatinib (HuH7: 0.8 µM, Hep3B: 0.4 µM). Malondialdehyde (MDA) levels and cell viability were analyzed. The data are representative of at least three independent experiments. *P < 0.01 versus the control group. (C) HCC cells were treated with ML385, and Nrf2, p‐Nrf2, and NQO1 protein levels were assayed via immunoblotting. HCC cells were treated with ML385 and lenvatinib (HuH7: 0.8 µM, Hep3B: 0.4 µM). Cell viability was analyzed using the CellTiter‐Glo assay. Hep3B and HuH7 cells were transfected with empty vector or Nrf2 plasmids, and Nrf2 protein levels were assayed via immunoblotting. The transfected cells were treated with lenvatinib (HuH7: 0.8 µM, Hep3B: 0.4 µM) or ferrostatin‐1 (fer1, 10 µM). MDA levels were analyzed. Cell viability was analyzed using the CellTiter‐Glo assay. (D) HCC cells transfected with Nrf2 overexpression or control plasmids were treated with lenvatinib (HuH7: 0.8 µM, Hep3B: 0.4 µM). Viability was analyzed using the CellTiter‐Glo assay. Len, lenvatinib

Fig 3: Enhanced NFE2L2 protein stability and activity during cuproptosis. (A, B) Western blot and qPCR analyses of the expression of NFE2L2 in PDAC cells in whole cell extracts after treatment with ES-Cu (100 nM, 1:1) for 6–24 h (n = 3 biologically independent samples, *P < 0.05 versus control, ANOVA test). (C) CHX chase assay of the degradation kinetics of NFE2L2 protein in PANC1 cells after treatment with CHX (50 µg/ml) in the absence or presence of 100 nM ES-Cu or 2 µM MG132 for 15–60 min (n = 3 biologically independent samples, *P < 0.05 versus CHX group, ANOVA test). ACTB was used as a loading control. (D) NFE2L2 transcriptional activity assay of PDAC cells following treatment with ES-Cu (100 nM) for 6–24 h (n = 3 biologically independent samples, *P < 0.05 versus control, ANOVA test). (E) Image analysis of the localization of NFE2L2 in PANC1 cells after treatment with ES-Cu for 24 h. Bar = 15 μm.

Fig 4: NFE2L2 mediates GCLM and GCLC expression during cuproptosis. (A) qPCR analysis of gene expression in PDAC cells following treatment with ES-Cu (100 nM) or erastin (10 µM) for 24 h. The data is displayed as a heatmap representing the mean of three biologically independent samples. (B) qPCR analysis of NFE2L2 gene expression in WT and NFE2L2-knockout (KO) cells (n = 3 biologically independent samples, *P < 0.05 versus WT group, ANOVA test). (C) qPCR analysis of gene expression in PDAC cells following treatment with ES-Cu for 24 h (n = 3 biologically independent samples, *P < 0.05 versus WT group, ANOVA test). (D) qPCR analysis of gene expression of NFE2L2, GCLM, or GCLC in indicated PANC1 cells following treatment with ES-Cu for 24 h (n = 3 biologically independent samples, *P < 0.05 versus KO group, ANOVA test). (E) Western blot analysis of protein expression in indicated WT and NFE2L2-KO PDAC cells after treatment with ES-Cu for 24 h.