Fig 2: Effects of crocin on Sirt1/Nrf2/HO-1 pathways in PHN rats. (A–D) The protein expression of Sirt1, Nrf2, and HO-1 were evaluated by western blotting. N = 3. Data are represented as mean ± SD from independent groups. **p < 0.01.

Fig 3: OIS-PRM analysis of FFPE oropharyngeal squamous cell carcinomas revealed a large dynamic range of NRF2 target expression.A, schema describing the collection and analysis of 20 HPV(−) and 10 HPV(+) oropharynx tumors. Protein was extracted from 50-micron curls cut from FFPE tumor blocks and subject to OIS-PRM. B, summary statistics for data-dependent acquisition proteomics on the 10 HPV(+) tumors lysine methylation and N-terminal lysine formylation are common artifacts of formalin fixation. C, PCA plot from protein abundances of differently expressed NRF2 target proteins as measured by OIS-PRM. D, scatterplot of NRF2 activity scores and SOX2 protein abundance for each tumor. SOX2 abundance is reported as the Log2-transformed geometric mean of the peak area ratios for the two representative peptides: APCQAGDLR and LLSETEK. The NRF2 score refers to the position along the first principal component from (C). E, heatmap of OIS-PRM data with row-normalized Z-scores. HPV, human papilloma virus; PCA, principal component analysis.

Fig 4: Combined analysis of three Clinical Proteomics Tumor Analysis Consortium cohorts showed that a strongly correlated subset of proteins could predict NRF2/KEAP1 genotype.A, protein expression of thirteen NRF2 targets in CPTAC cohorts for HNSCC, LSCC, and LUAD. NRF2 mutations distant from the ‘DLG’ or ‘ETGE’ motifs were excluded from the count. B, PCA plot of the first two principal components for the CPTAC data by the thirteen NRF2 target peptides. The NRF2 activity score refers to the position along the first principal component of the data. C, distribution of NRF2 scores for the combined CPTAC cohorts split based on the presence or absence of missense mutations in KEAP1 or NRF2. D, a LASSO regression model trained to predict the NRF2 scores for tumors given the 6133 proteins not included in the original set of thirteen NRF2 targets. The model was trained by 10-fold cross validation on a training set of 2/3 of the data (218 tumors). Predicted NRF2 scores are plotted against the true NRF2 pathway scores for an independent test set containing one-third of the data (109 tumors). E, feature weights for the regression model in (C). F, protein to mRNA correlations for the NRF2 IS-PRM panel proteins across the cancer cell line encyclopedia. CPTAC, Clinical Proteomic Tumor Analysis Consortium; HNSCC, head and neck squamous cell carcinoma; IS-PRM, internal standard–triggered parallel reaction monitoring; PCA, principal component analysis.

Fig 5: 5-fluorouracil disturbs the antioxidative/oxidative balance, resulting in CD3+ T cells ferroptosis. A: Levels of antioxidant proteins nuclear erythroid 2 p45-related factor 2, heme oxygenase-1, and glutathione peroxidase 4 assessed by western blot, and corresponding graphs showing the statistical comparison between groups; B and C: Reactive oxygen species (ROS) and lipid ROS of rat and human CD3+ T cells were detected by FACS using DCFH-DA and C11-BODIPY fluorescent conjugates, BODIPY emission was recorded on oxidized C11 (FITC channel) signal; the graphs display the comparison of mean fluorescence intensity levels in the different groups; D: Proportion of cells with reduced mitochondrial membrane potential assessed by flow cytometry; E and F: Glutathione and malondialdehyde content T cells after treatment for 48 h. CON groups: T cells were treated with DMSO for 48 h; 5-fluorouracil (5-FU) groups: T cells were treated with 5-FU (15 μmol/L) for 48 h; 5-FU+ferrostatin-1 (Fer-1) groups: T cells were treated with 5-FU (15 μmol/L) and Fer-1 (2 μmol/L) for 48 h. Statistical analysis was done by one-way ANOVA, n = 3. dP < 0.05 vs the 5-FU group, eP < 0.01 vs the 5-FU group, fP < 0.001 vs the 5-FU group. Nrf2: Nuclear erythroid 2 p45-related factor 2; HO-1: Heme oxygenase-1; GPX4: Glutathione peroxidase 4; GSH: Glutathione; MDA: Malondialdehyde; CON: Untreated control groups; 5-FU: 5-fluorouracil; Fer-1: Ferrostatin-1; MFI: Mean fluorescence intensity.