Fig 2: Keap1 knockdown facilitated A549 and H1299 cell lines proliferation.(A) Representative images of colony formation assay in A549 and H1299 cell lines expressing different levels of Keap1 were shown. (B) Quantitative analysis of total colony numbers in A549 and H1299 cells was shown. (C) Cell proliferation abilities in A549 and H1299 cell lines expressing different levels of Keap1 were analysed by cell growth curves. Statistical significance was tested by unpaired t test. Values were given as mean ± SD. **P < 0.01, ***P < 0.001 compared to the control group.

Fig 3: Effect of NaB on neuroinflammatory molecules and Nrf2/HO-1 signaling in LPS-stimulated primary microglia. Primary microglia were incubated with LPS, LPS+NaB, or LPS+NaB+ML385 for 12 hours. (A and B) Immunofluorescence images of cultured primary microglia showing expressions of IBA-1 (green), iNOS (red) or COX-2 (red) and DAPI (blue) at 12 h after LPS or LPS+NaB treatment when compared with the corresponding control. (C) NaB treatment inhibits the LPS-induced ROS production in primary microglia. Cells were analyzed with microplate reader. (D) Western blot analysis of Nrf2, Keap1, HO-1, NQO1, iNOS, COX-2, and BDNF protein expressions in the primary microglia at 12h after LPS, LPS+NaB, and LPS+NaB+ML385 and their corresponding controls. GAPDH served as the loading control. Bar graphs depicting the optical density of Nrf2, Keap1, HO-1, NQO1, iNOS, COX-2, and BDNF expressions. Data presented as mean ± SD (n = 3 per group). Scale bars: 20µm. *P < 0.05, **P < 0.01 by one-way ANOVA.

Fig 4: EBV infection activates the p62-Keap1-NRF2 signaling pathway and induces high GPX4 expression in NPC cells.mRNA and protein expression levels of GPX4 and SLC7A11 in EBV-negative and EBV-positive NPC cells were determined by RT–qPCR (A) (n = 3) and immunoblotting (B) (n = 3). C The p62-Keap1-NRF2 pathway was examined in EBV-negative and EBV-positive NPC cells by immunoblotting. D Cytoplasmic and nuclear proteins from EBV-negative and EBV-positive NPC cells were fractionated and detected by immunoblotting. Lamin B1 and GAPDH were used as controls for the nuclear and cytoplasmic fractions, respectively. E Immunofluorescence staining showing the localization of NRF2 in EBV-negative and EBV-positive NPC cells. mRNA and protein levels of NRF2 and GPX4 in EBV-negative and EBV-positive NPC cells were determined by RT–qPCR (F) (n = 3) and immunoblotting (G) (n = 3) after siRNA knockdown of endogenous NRF2. H GPX4 was highly expressed in CNE2 EBV + xenografts. I Representative images of immunohistochemistry staining showing GPX4 expression in paraffin-embedded tumor sections from NPC patients. J GPX4 expression in different groups according to EBV copy number in 181 NPC patients. Data are shown as the mean ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. A, F Two-tailed unpaired t test (F, compared to si-NC). J two-tailed Mann–Whitney test. Scale bars: 20 µm (E) and 100 µm (H, I).

Fig 5: Keap1 knockdown facilitated EMT of A549 and H1299 cell lines.(A) Western blot analysis of epithelial markers E-cadherin, mesenchymal markers (N-cadherin, Vimentin) and Snail in A549 cells with different Keap1 expression levels. (B) Quantitative analysis of EMT protein in A549 cell line was shown. (C) Western blot analysis of epithelial markers E-cadherin, mesenchymal markers (N-cadherin, Vimentin) and Snail in H1299 cells with different Keap1 expression levels. (D) Quantitative analysis of EMT protein in H1299 cell line was shown. Statistical significance was tested by unpaired t test. Values were given as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group.