Fig 2: Keap1 forms oxidative stress-induced inclusions in breast cancer cell lines. (A,B) Endogenous Keap1 expression in two breast cancer cell lines, 21MT-1 (A) and SKBR3 (B), treated with 300 µM H2O2 for 3 h, visualized by immunofluorescence microscopy. (C) Quantification of Keap1 inclusions following hydrogen peroxide treatment in (A,B). Means derived from three biological replicates were used during the analysis. Means were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Data are expressed as mean ± SD. p < 0.05 was considered statistically significant; ** p < 0.01, *** p < 0.001.

Fig 3: Cold exposure induces oxidative stress in the colon and regulates SIRT1/Nrf2-and SIRT1/NF-?B-mediated pathways in vivo. Effects of cold exposure on SOD (A), GSH (B) and MDA (C) levels in the mouse colon. The protein expression levels (D) and enzyme activity (F) of SIRT1. (D) The protein expression levels of total-Nrf2,Keap1, nuclear-Nrf2, NQO1, HO-1, p65, and acetylated-p65 were measured by western blot analysis in mouse colon tissue. (E) The acetylation level of Nrf2 was determined using co-immunoprecipitation assays in mouse colon tissue. Data are presented as the mean ± SEM (n = 3). Statistically significant differences are indicated; *p < 0.05, **p < 0.01, ***p < 0.001, significantly different from control group.

Fig 4: Effect of Met on the expression level of genes and proteins associated with the Nrf2/Keap1 signaling pathway in the cardiac tissue of the three experimental groups. (A) mRNA levels of NFE2L2, (B) KEAP1 and (C) HMOX-1. (D) Representative western blot images of proteins associated with the Nrf2/Keap1 signaling pathway. (E) Densitometry analysis of Nrf2, (F) Keap1 and (G) HO-1 expression. The relative densitometry is expressed as the ratio of Nrf2, Keap1 or HO-1 to GAPDH. Data are presented as mean ± SEM; n=6 per group. **P<0.01 vs. Control; ?P<0.05 vs. HFD. HFD, high fat diet; HO-1, heme oxygenase 1; Keap1, kelch-like ECH-associated protein 1; HMOX-1, HO-1 gene; Met, metformin; Nrf2, nuclear factor (erythroid-derived 2)-like 2; NFE2L2, Nrf2 gene.

Fig 5: K67, a chemical compound that inhibits the interaction between Keap1 and S349-phosphorylated p62.(a) Working hypothesis about an inhibitors of the interaction between Keap1 and S349-phosphorylated p62. (b) Structural formulas of K67 and Cpd16. (c) Crystal structure of Keap1-DC (white) in complex with K67 (cyan). Overall structure of Keap1-DC, shown as a ribbon model. K67 (cyan) and some of the potential interacting residues of Keap1-DC are shown in stick representation. A close-up view of K67 bound to Keap1-DC is shown. Intermolecular electrostatic interactions are depicted as broken red lines. (d) In vitro pull-down assay. Full-length Keap1 was allowed to form a complex with K67 (left panel) or Cdp16 (right panel) at the indicated concentration. Upper panel: Keap1 binding to His-GST-tagged Neh2(?17-51) was estimated by Oriole fluorescent gel staining. Middle and bottom panels: Keap1 binding to OSF-p-p62 (middle panel) or OSF-Nrf2 (bottom panel) was estimated by immunoblot analysis with anti-Keap1 antibody. Data are representative of three independent experiments. (e) Modelling of Keap1 (white)–K67 (cyan)–DLGex (green; upper left panel) and Keap1–Cpd16 (orange)–DLGex (lower left panel). Upper right: schematic representation of the interface between DLGex and K67. Lower right: schematic representation of the interface between DLGex and Cpd16. Potential hydrogen bonds are indicated by dashed lines between the atoms involved, whereas hydrophobic contacts are represented by an arc with spokes radiating towards the ligand atoms they contact. (f) In vitro pull-down assay. Full-length Keap1 was allowed to form a complex with K67 (left panel) or Cdp16 (right panel) at the indicated concentration. Keap1 binding to His-GST-tagged Neh2(1-56) was estimated by Oriole fluorescent gel staining. Data are representative of three independent experiments. (g) In vitro pull-down assay with biotinylated compounds. His-GST-tagged Neh2(1-56) binding to the Keap1-DC complexed with biotinylated K67 or Cpd16 was estimated by Coomassie brilliant blue staining. Data are representative of three independent experiments.