Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: Transcriptomic and molecular analysis of the potential pathways involved in MMBOx-mediated BMSCs rejuvenation. (A) Circular heatmap showing differentially expressed genes (DEGs) associated with cell senescence, inflammation, and osteogenesis in senescent BMSCs treated with MMBOx@GPP compared to GPP. (B) Gene Ontology (GO) enrichment analysis of upregulated DEGs. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs. (D) Gene Set Enrichment Analysis (GSEA) plots of the glutathione metabolic process (ES: enrichment score; NES: normalized enrichment score; FDR: false discovery rate). (E) Heatmap of DEGs enriched in aging-related GO terms. (F) Western blot analysis of Keap1, Nrf2, Nqo1, Gclc, and GAPDH protein expression in BMSCs. (G) Quantitative analysis of protein band intensities ( n = 3). (H) Representative flow cytometry plots of ThiolTracker™ fluorescence staining indicating intracellular glutathione levels. (I) Quantification of intracellular GSH/GSSG ratio in BMSCs ( n = 3). (J) Schematic diagram illustrating the proposed mechanism by which MMBOx attenuates BMSCs senescence via Nrf2 pathway activation and glutathione metabolism enhancement. Data are expressed as mean ± SD; ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Western blotting was employed to evaluate protein expression of key targets, including Keap1 (Affinity, AF5266; 1:1000), Nrf2 (Proteintech, 16396-1-AP; 1:1000), Nqo1 (Abcam, ab80588; 1:10,000), Gclc (Proteintech, 12601-1-AP; 1:25000) and GAPDH (Proteintech, 60004-1-Ig; 1:50,000).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Staining, Activation Assay

Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: Transcriptomic and molecular analysis of the potential pathways involved in MMBOx-mediated BMSCs rejuvenation. (A) Circular heatmap showing differentially expressed genes (DEGs) associated with cell senescence, inflammation, and osteogenesis in senescent BMSCs treated with MMBOx@GPP compared to GPP. (B) Gene Ontology (GO) enrichment analysis of upregulated DEGs. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs. (D) Gene Set Enrichment Analysis (GSEA) plots of the glutathione metabolic process (ES: enrichment score; NES: normalized enrichment score; FDR: false discovery rate). (E) Heatmap of DEGs enriched in aging-related GO terms. (F) Western blot analysis of Keap1, Nrf2, Nqo1, Gclc, and GAPDH protein expression in BMSCs. (G) Quantitative analysis of protein band intensities ( n = 3). (H) Representative flow cytometry plots of ThiolTracker™ fluorescence staining indicating intracellular glutathione levels. (I) Quantification of intracellular GSH/GSSG ratio in BMSCs ( n = 3). (J) Schematic diagram illustrating the proposed mechanism by which MMBOx attenuates BMSCs senescence via Nrf2 pathway activation and glutathione metabolism enhancement. Data are expressed as mean ± SD; ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Western blotting was employed to evaluate protein expression of key targets, including Keap1 (Affinity, AF5266; 1:1000), Nrf2 (Proteintech, 16396-1-AP; 1:1000), Nqo1 (Abcam, ab80588; 1:10,000), Gclc (Proteintech, 12601-1-AP; 1:25000) and GAPDH (Proteintech, 60004-1-Ig; 1:50,000).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Staining, Activation Assay

Journal: Molecules

Article Title: Subcritical Water Extract from Grape Pomace Protects Human Bronchial Epithelium Cells by Mitigating Oxidative Stress Through Nrf2 Pathway

doi: 10.3390/molecules31101736

Figure Lengend Snippet: SWE 160.1 treatment increased the antioxidant response. ( a ) Western blotting analysis of antioxidant factors (HO-1, SOD-2 and Nrf2) after 48 h of treatment with 75 and 100 µg GAE/mL SWE 160.1 in HBE cells. Protein levels were normalized to γ-tubulin. ( b ) qRT-PCR analysis of Nrf2, Keap1 and HO-1 mRNA levels after 24 h of treatment with 75 and 100 µg GAE/mL SWE 160.1 in HBE cells. Gene expression levels represent the relative mRNA expression compared to the untreated cells, normalized to GAPDH mRNA. ( c ) Western blotting of subcellular fractions of control and 48 h SWE 160.1 -treated cells incubated with pNrf2 (Ser40) antibody. Lamin A and GADPH antibodies marked as nuclei (N) and cytoplasmic (C) fractions, respectively. The images are representative of three different experiments. * p < 0.05, ** p < 0.01 vs. untreated control cells.

Article Snippet: The primers used were GAPDH (Proligo USA, Milan, Italy), Nrf2 ( HP209154 , OriGene Technologies, Inc., Rockville, MD, USA) and HO-1 ( HP205872 , OriGene Technologies, Inc., USA).

Techniques: Western Blot, Quantitative RT-PCR, Gene Expression, Expressing, Control, Incubation

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: Regulation of lipid peroxidation is extrinsically regulated by the SLC7A11 pathway in CD4 + T cells (A) Statistical analysis of SLC7A11 protein expression assessed by flow cytometry (shown as mean fluorescent intensity (MFI). CD4 + T cells were either left unstimulated (control) or measured at a distinct time point after stimulation with anti-CD3/CD28 antibodies. N = 3, 3 independently performed experiments, a one-way ANOVA multiple comparison test was performed. The calculated power of this experiment is 0.84. (B) ΔCT of SLC7A11 mRNA expression in human CD4 + T cells with and without anti-CD3/CD28 stimulation. N = 13, 4 independently performed experiments. (C) Gene expression in CD4 + T cells from WT and VAV cre Keap fl/fl (Keap1-KO) mice was assessed by microarray analysis. Colors indicate significant upregulation (red), or downregulation (green) compared to WT. (D) ΔCT of SLC7A11 mRNA expression in CD4 + T cells of WT and Keap1-KO mice, either left unstimulated or stimulated with anti-CD3/CD28 antibodies, 4 independently performed experiments. The calculated power of this experiment is 0.99 for the stimulated and unstimulated groups. (E) N-fold SLC7A11 expression in human CD4 T cells, either transfected with a control siRNA (Ctrl) or NRF2 siRNA. 3 independently performed experiments were performed. (F) N-fold SLC7A11 mRNA expression in human CD4 T cells treated with 4-OI or vehicle (Ctrl) N = 6, 4 independently performed experiments. A paired Student's t-test was performed comparing the groups of B–F. Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: To this, 10 nM NRF2 siRNA (OriGene, Germany; catalogue number SR321100) or a control was added, and transfection was performed according to the manufacturer’s instructions (Neon transfection, ThermoFisher Scientific; MPK10025).

Techniques: Expressing, Flow Cytometry, Control, Comparison, Gene Expression, Microarray, Transfection

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: SAS and erastin treatment reduce cystine uptake but not GSH content and cell viability in anti-CD3/CD28 stimulated CD4 + T cells (A) Flow cytometric analysis of cystine uptake measured by BioTracker Cystine-FITC in stimulated CD4 + T cells in the presence of SAS and erastin. Exemplary histogram, showing the cystine uptake after 0 min (blue) and after 30 min (red) of incubation, while the difference presented as ΔMFI was used to describe the cystine uptake. N = 4, 3 independently performed experiments. The calculated power of this experiment is 0.99. (B) Statistical analysis of cystine uptake (represented as MFI) of mouse WT and NRF2-KO CD4 + T cells with and without the addition of SAS and erastin. N = 6, 4 independently performed experiments. (C) Representative histograms of B showing the uptake after 0 min (blue) and 30 min (red), while the difference presented as ΔMFI was used to describe the cystine uptake. (D) Flow cytometric analysis of reactive oxygen species (ROS) (presented as MFI) of CD4 + T cells without and with the addition of SAS and erastin. Histograms of the measurements are shown on the right. The calculated power of this experiment is 0.986. (E) Flow cytometric analysis of intracellular GSH content (presented as ΔMFI) of CD4 + T cells with and without SAS and erastin. Histograms depict an overlay of the FMO (blue) and the GSH staining (red). GSH was determined by the GSH/GSSG Luminescence measurement of CD4 MACS HC PBMCs. The kit provides direct analysis of the supernatants' GSH and GSSG contents, which enables the calculation of the ratios. (F) Lipoperoxidation measurement using BODIPY 581/591 C11 (presented as MFI of the FITC signal) of CD4 + T cells without and with inhibition by SAS and erastin. Histograms of the two signals, i.e., PE and FITC, from BODIPY are shown on the right. The calculated power of this experiment is 0.94. (G) Flow cytometric analysis of Mitotracker (represented as MFI, mitochondrial mass) of CD4 + T cells and TMRM (represented by MFI, mitochondrial membrane potential) of Mitotracker positive cells is shown without and with the addition of SAS and erastin. The calculated power of this experiment is 0.99 for the mitochondrial mass and 1 for the mitochondrial membrane potential. (H) Fluorescent live/dead staining was evaluated by flow cytometry using the combinatory staining of Annexin V and fixable viability dye (FVD) without and with the addition of SAS and erastin. N = 3, 3 independent experiments were performed for the graphs (D) to (G). The statistical evaluation of all the represented graphs was performed using one-way ANOVA multiple comparison. (Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: To this, 10 nM NRF2 siRNA (OriGene, Germany; catalogue number SR321100) or a control was added, and transfection was performed according to the manufacturer’s instructions (Neon transfection, ThermoFisher Scientific; MPK10025).

Techniques: Incubation, Staining, Inhibition, Membrane, Flow Cytometry, Comparison

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: Characterization of an altered NRF2/cystine pathway in patients with JIA (A) Flow cytometric analysis of SLC7A11 expression in stimulated SF and PB CD4 + T cells. Statistical analysis was performed with N = 7 donors, 7 independently performed experiments. (B) Flow cytometric evaluation of cystine uptake (shown as MFI) by anti-CD3/CD28 stimulated CD4 + SF T cells. N = 6, 6 independently performed experiments. (C) Flow cytometric analysis of intracellular glutathione content (presented as ΔMFI) of stimulated SF and PB CD4 + T cells. N = 9, 9 independently performed experiments. (D) Statistical analysis of MFI of ROS in stimulated SF and PB CD4 + T cells ( N = 4), 4 independently performed experiments. The calculated power of this experiment is 0.85. (E) Lipid peroxidation as assessed by flow cytometric measurement of anti-CD3/CD28 stimulated CD4 + T cells derived from PBMCs and SFMCs. N = 11. (F) Gene set enrichment analysis (GSEA) was performed on differentially expressed genes from SF-derived CD4 + T cells of patients with active JIA compared to PBMC-derived CD4 + T cells from healthy controls. The enrichment plots shown represent driver ferroptosis-related gene sets from KEGG, WikiPathways, and FerrDb V2. ( N = 4). (G) Heat maps showing normalized counts of selected RNAs determined by RNA-seq in JIA CD4 + T cells compared to HC CD4 + T cells. N = 4 in each group. (H) SFMCs and PBMCs were analyzed for ASCT1 expression by quantitative RT-PCR. The calculated power of this experiment is 0.99. (I) Flow cytometric analysis of CD36 expression of stimulated SF and PB CD4 + T cells. N = 5, 5 independently performed experiments. (J) Representative histograms showing BODIPY Ferroptosis staining of anti-CD3/CD28 stimulated HC PBMCs in the absence and presence of ASCT1/2 inhibition by 4-Hydroxy-L-phenylglycin (HPG) and SAS. N = 3, 3 independent experiments. The statistical evaluation of (A), (C), (E), (H), and (I) from the represented graphs was performed using a paired Student’s t-test. One-way ANOVA multiple comparison was used for (B) and (D) of the represented graphs. Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: To this, 10 nM NRF2 siRNA (OriGene, Germany; catalogue number SR321100) or a control was added, and transfection was performed according to the manufacturer’s instructions (Neon transfection, ThermoFisher Scientific; MPK10025).

Techniques: Expressing, Derivative Assay, RNA Sequencing, Quantitative RT-PCR, Staining, Inhibition, Comparison

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation upregulates SLC7A11 through AMPK-NRF2 axis. ( A ) Western blot analysis of lysates from NC and SLC31A1 knockdown (shRNA#2) AsPC-1 cells transfected with non-targeting scrambled siRNA (siNC) or AMPK siRNA (siAMPKα1/2). ( B ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNFE2L2#2). * indicates a non-specific band. ( C ) SLC7A11 mRNA level in NC and SLC31A1 knockdown AsPC-1 cells transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2#2). ( D ) Western blot analysis of lysates from AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( E ) SLC7A11 mRNA level in AsPC-1 cells that were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2) and treated with TM (50 μM) or without TM for 24 h. ( F ) Western blot analysis of lysates from NC and SLC31A1 knockdown AsPC-1 cells treated with or without ML385 (5 μM) for 24 h. ( G ) Western blot analysis of lysates from SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. ( H ) SLC7A11 mRNA level in SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid. Mean ± SD, n = 3. Statistical significance was determined using a one-way ANOVA test.

Article Snippet: NFE2L2/NRF2 S558A CDNA (with Flag) , Shanghai Genechem , This paper.

Techniques: Western Blot, Knockdown, shRNA, Transfection, Plasmid Preparation

Journal: Redox Biology

Article Title: Copper deprivation reprograms antioxidant defense to suppress ferroptosis via SLC7A11

doi: 10.1016/j.redox.2026.104130

Figure Lengend Snippet: Copper deprivation inhibits ferroptosis by the activation of AMPK-NRF2-SLC7A11 pathway. ( A ) NC and SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( B ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). PI staining images of the indicated AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of TM (50 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( C ) SLC31A1 knockdown AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA#2 (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) for 2 h. ( D ) AsPC-1 cells were transfected with non-targeting scrambled shRNA (shNC) or NRF2 shRNA (shNRF2). Lipid peroxidation of the indicated AsPC-1 cells treated with RSL3 (0.25 μM) in the presence or absence of TM (50 μM) for 2 h. ( E ) PI staining images of the NC and SLC31A1 knockdown AsPC-1 cells treated with RSL3 at the indicated concentrations in the presence or absence of ML385 (5 μM) for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm. ( F ) PI staining of SLC31A1 knockdown (shRNA#2) and NRF2 knockdown (shRNA#2) AsPC-1 cells transfected with a blank vector or NRF2 WT or NRF2 S558A plasmid, followed by treatment with RSL3 at the indicated concentrations for 6 h. Quantification of PI-positive cells was shown. Mean ± SD, n = 3. Statistical significance was determined using a two-way ANOVA test. Scale bar: 50 μm.

Article Snippet: NFE2L2/NRF2 S558A CDNA (with Flag) , Shanghai Genechem , This paper.

Techniques: Activation Assay, Knockdown, Transfection, shRNA, Staining, Plasmid Preparation