Journal: Frontiers in Physiology

Article Title: The possible mechanisms linking chronic obstructive pulmonary disease and coronary atherosclerosis based on coronary computed tomography angiography and animal experiments

doi: 10.3389/fphys.2026.1688832

Figure Lengend Snippet: Oxidative stress response and the KEAP1–NRF2 pathway levels are linked to CAS in COPD patients. (A) ELISA detection of KEAP1 and NRF2 levels in the serum of patients. (B) KEAP1, NRF2, NQO1, and HO-1 levels in the serum of patients determined via RT-qPCR. (C) Levels of ROS and MDA, and activities of SOD and catalase in the serum of patients determined using kits. The CAS group was used as a normalization control. Data among multiple groups were compared using one-way ANOVA, followed by Tukey’s post-hoc tests with corrections for multiple comparisons. CAS, coronary atherosclerosis; COPD, chronic obstructive pulmonary disease; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase.

Article Snippet: Serum KEAP1 and Nrf2 levels were measured using a human KEAP1 enzyme-linked immunosorbent assay (ELISA) kit (EH4240, Wuhan Fine Biotech Co., Ltd., Wuhan, Hubei, China) and a human Nrf2 ELISA kit (abs551899, Absin Bioscience Inc., Shanghai, China) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control

Journal: Frontiers in Physiology

Article Title: The possible mechanisms linking chronic obstructive pulmonary disease and coronary atherosclerosis based on coronary computed tomography angiography and animal experiments

doi: 10.3389/fphys.2026.1688832

Figure Lengend Snippet: KEAP1–NRF2-mediated oxidative stress participates in the occurrence of COPD combined with CAS in mice. (A) PFT for FRC, RI, Cdyn, and MV in mice. (B) The histopathological changes of lung tissues observed using H&E staining. (C) Serum lipid levels in mice measured via ELISA. (D) KEAP1, NRF2, NQO1, and HO-1 levels in the serum of mice determined via RT-qPCR. (E) Levels of ROS and MDA, and activities of SOD and catalase in the lung tissue of mice measured using kits. (F) Representative Western blotting of KEAP1, NRF2, NQO1, and HO-1 proteins in the lung tissue of mice and the quantification data. n = 6 mice for each treatment. Data among multiple groups were compared using one-way ANOVA, followed by Tukey’s post-hoc tests for multiple comparisons. COPD, chronic obstructive pulmonary disease; CAS, coronary atherosclerosis; PFT, pulmonary function test; FRC, functional residual capacity; RI, resistance index; Cdyn, dynamic compliance; MV, minute ventilation; ROS, reactive oxygen species; MDA, malondialdehyde; SOD, superoxide dismutase.

Article Snippet: Serum KEAP1 and Nrf2 levels were measured using a human KEAP1 enzyme-linked immunosorbent assay (ELISA) kit (EH4240, Wuhan Fine Biotech Co., Ltd., Wuhan, Hubei, China) and a human Nrf2 ELISA kit (abs551899, Absin Bioscience Inc., Shanghai, China) according to the manufacturer’s instructions.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Functional Assay

Journal: Redox Biology

Article Title: N-acetyl- l -cysteine ethyl ester (NACET) induces the transcription factor NRF2 and prevents retinal aging and diabetic retinopathy

doi: 10.1016/j.redox.2025.103914

Figure Lengend Snippet: NACET increases intracellular levels of Cys, which directly conjugate to Cys residues 226 and 613 of KEAP1 and induce NRF2 expression. a , Western blot analysis of KEAP1 and NRF2 expression in ARPE-19 cells following lentiviral-mediated expression of shRNAs targeting 3′UTR of KEAP1 mRNA (shKEAP1) or GFP (Ctrl). Where indicated. Cells were treated with 1 mM NACET for 2 h b , Schematic representation of the GSH synthesis pathway. The enzymes involved are boxed; abbreviations: ACY1, aminoacylase 1; GCL, glutamate-cysteine ligase; GSS, glutathione synthetase. c , Heatmap showing the status of sensor cysteine residues identified by mass spectrometry analysis of purified 3xFLAG-KEAP1 transfected into HEK 293 cells treated and not treated with NACET: unconjugated (purple squares), NACET-, NAC-, Cys- or GSH-conjugated in NACET-treated and untreated samples (cyan squares) or only in NACET-treated samples (yellow squares). d , AlphaFold model showing the domains of KEAP1 and the relative position of the 12 Cys residues found conjugated in the MS analysis. e , Scheme of the transfection protocol for the analysis of NRF2 transactivation activity. pRL-SV40 was used for the constitutive expression of Renilla Luciferase for normalization. f , NRF2 activity measured as transactivation of the ARE-luc reporter gene in cells silenced for endogenous KEAP1 expression. KEAP1 silenced cells were transfected with reporter plasmids and one plasmid for the expression of shRNA-resistant KEAP1 (wt or mutant, as indicated). 8 h post transfection, the cells were grown for 24 h in growth medium ±0.2 mM NACET. KEAP1 Cys residues were substituted with serine, glutamic acid (C288E) or tryptophan (C273W) as described by Suzuki et al. . Values represent the fold change in normalized luciferase activity in cells transfected for the expression of the corresponding KEAP1 variant and are expressed as means ± SD (N = 3–5). Differences were tested using one-way ANOVA followed by Dunnett's multiple comparisons test. g, Confluent ARPE-19 cells were pre-treated for 30 min with 100 μM BSO where indicated and then incubated with 0.2 mM NACET for the indicated time points, lysed by TCA-EDTA solution and the intracellular thiols were measured by HPLC. Thiol concentrations are normalized for the total protein content and expressed in nmol/mg protein. Measurements are the means ± SD of 4 independent experiments. Differences were tested using one-way ANOVA followed by Tukey's multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. For clarity, not all the significant differences are indicated. h , Confluent ARPE-19 cells were incubated with 1 mM NAC for 1 h, and thiols were extracted and measured as in ( g ). i , Whole lysates of cells treated as in ( g ) were analyzed by Western blot with anti-NRF2 antibody. Anti-GAPDH antibody was used as a loading control. Densitometric analysis of bands is shown. j , Cartoon showing the proposed NACET's tripartite mechanism of action.

Article Snippet: Plasmid for KEAP1 ( NM_012289 ) expression in KEAP1-silenced cells was purchased from ORIGENE (#SC111946; Rockville, MD, USA).

Techniques: Expressing, Western Blot, Mass Spectrometry, Purification, Transfection, Activity Assay, Luciferase, Plasmid Preparation, shRNA, Mutagenesis, Variant Assay, Incubation, Control