ZVI-NP inhibited NRF2-regulated antioxidant activity via enhancement of GSK3β/β-TrCP degradation pathway. A, Immunoblotting of NRF2 and GPX4 in four cancer cell lines treated with ZVI@CMC NPs at the indicated doses for 24 h. GAPDH was used as internal control. B, ChIP-qPCR assay was performed to measure NRF2 binding ability to the promoter region of SLC7A11, AKR1C1 and AIFM2 in cells treated with ZVI@Ag NPs in A549 (upper) and H460 (lower). C, mRNA expression of NRF2 downstream genes was measured by RT-qPCR after ZVI@Ag NPs treatment in A549 and H460 cells. The heat maps reflect downregulation of the mRNA levels of these genes compared to the respective untreated controls. D, Immunoblotting of p-AMPK, total AMPK, p-mTOR, total mTOR, p-GSK3β and total GSK3β in cells treated with ZVI@Ag NPs (left) or ZVI@CMC NPs (right) for the indicated time. E, Immunofluorescence staining of NRF2, β-TrCP and DAPI in A549 (left) and H460 (right) cells after ZVI@Ag NPs or ZVI@CMC NPs treatment. Data were mean ± s.e.m. (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.