Journal: Redox Biology

Article Title: Amino acid restriction sensitizes lung cancer cells to ferroptosis via GCN2-dependent activation of the integrated stress response

doi: 10.1016/j.redox.2025.103988

Figure Lengend Snippet: Increased mitochondrial respiration renders F12-cultured cells BSO-sensitive. ( A, B ) Oxygen consumption rate (left) in F12 or F12AA-cultured A549 (A) or H838 (B) cells treated with 0.5 μM oligomycin (Oligo), 1 μM FCCP, and 0.5 μM rotenone (Rot), as indicated. Graphs (right) showing parameter data extracted from the oxygen consumption rate and extracellular acidification rate (n = 14 in A549 cells; n = 10 in F12 and 4 in F12AA-cultured H838 cells). ( C , D ) Oxygen consumption rate (left) in F12-cultured A549 (C) or H838 (D) cells transfected with control siRNA or siRNA targeting ATF4 mRNA and assayed as in (A, B). Graphs (right) show extracted parameters (n = 15 for A549; n = 21 for ATF4 siRNA and n = 13 for control siRNA in H838). ( E ) MitoSox fluorescence images of A549 (left) and H838 (right) cells cultured in F12 or F12AA medium. Cells were treated with 30 μM BSO for 40 h (A549) or 48 h (H838). Graphs show IncuCyte-based fluorescence quantification over time. Scale bar, 100 μm. ( F ) MitoSox fluorescence images of A549 cells cultured in F12 or F12AA medium and treated for 24h with 50 μM BSO, 50 μM BSO + 20 μM mito-TEMPO (MT), or control. MitoSox was added during the last 90 min of treatment; cells were then washed and quantified by live imaging. The graph shows IncuCyte-based fluorescence quantification. Scale bar, 100 μm; n = 8–10. ( G ) Confocal microscopy images of F12-cultured A549 cells treated with 100 μM BSO for 24h showing reduced (pink) and oxidized (green) BODIPY-C11 in combination with mitotracker deep red (red). Corresponding graphs show pixel-wise colocalization (Mander's coefficient) of oxidized BODIPY-C11 and mitotracker deep red in F12-cultured A549 (left) or H838 (right) cells treated with 100 μM BSO for 24 h or controls. n = 6–9 visual fields in A549 cells and 48 visual fields in H838 cells. Scale bar, 10 μm. ( H ) Oxidized BODIPY-C11 fluorescence images of A549 cells cultured in F12 medium and treated for 12 h with 100 μM BSO, 100 nM rotenone, 100 nM oligomycin, or combinations of BSO + rotenone or BSO + oligomycin, and controls. Graph shows IncuCyte-based fluorescence quantification. Scale bar, 50 μm. ( I ) Oxidized BODIPY-C11 fluorescence images of A549 cells cultured in F12 medium and treated for 24 h with 100 μM BSO, BSO + 20 μM mito-TEMPO (MT), or control. Graph shows IncuCyte-based fluorescence quantification. Scale bar, 100 μm. ( J ) Oxidized MitoPerOx fluorescence images of A549 (left) and H838 (right) cells cultured in F12 or F12AA medium. Cells were treated with 30 μM BSO or control for 48 h. Graphs show IncuCyte-based fluorescence quantification over time. Scale bar, 100 μm. ( K) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 100 nM oligomycin, 100 nM rotenone, 20 μM mito-TEMPO + rotenone, or mito-TEMPO + oligomycin, or control for 72 h. ( L ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 100 nM oligomycin, 100 nM rotenone, 5 μM ferrostatin-1 (FER) + rotenone, 5 μM liproxstatin-1 (LIP) + rotenone, ferrostatin-1 + oligomycin, or liproxstatin-1 + oligomycin, or control for 72 h. ( M ) Dose response curves of F12-cultured A549 cells treated with BSO in combination with 0.5 μM FCCP, or control for 72 h. ( N ) Dose response curves for F12-cultured A549 or H838 cells treated with BSO in combination with 25 or 50 μM mito-TEMPO, or control for 72 h. Dose response curves were normalized against the mean of the untreated samples for each condition. n = 3 replicates for all datapoints unless otherwise indicated. Error bars show SEM. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.

Article Snippet: Drugs used were auranofin (A6733; Sigma-Aldrich), α-tocopherol (T3251; Sigma-Aldrich), bafilomycin A1 (SML1661; Sigma-Aldrich), certolizumab pegol (HYP9953; MedChemExpress), chloroquine (C6628; Sigma-Aldrich), CU-CPT4A (HY-108473; MedChemExpress), deferoxamine (D9533; Sigma-Aldrich), erastin (e7781; Sigma Aldrich), FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) (HY-100410; MedChemExpress), ferrostatin-1 (SML0583; Sigma-Aldrich), l -buthionine-sulfoximine (b2515; Sigma-Aldrich), mito-TEMPO (HY–W001187; MedChemExpress), necrostatin-1 (N9037; Sigma-Aldrich), liproxstatin-1 (SML1414; Sigma-Aldrich), oligomycin (HY–N6782; MedChemExpress), puromycin dihydrochloride (A1113803; Gibco), rotenone (HY–B1756; MedChemExpress), RSL3 (SML2234; Sigma-Aldrich), resatorvid (HY-11109; MedChemExpress), and Z-VAD-FMK (V116; Sigma-Aldrich).

Techniques: Cell Culture, Transfection, Control, Fluorescence, Imaging, Confocal Microscopy

Journal: Biomedical Reports

Article Title: Mitochondrial oxidative stress under hypoxia promotes gastric mucosal injury in portal hypertensive gastropathy

doi: 10.3892/br.2026.2102

Figure Lengend Snippet: ROS scavenger Mito-TEMPO attenuates gastric mucosal epithelial injury from in vivo and in vitro models. (A) H&E staining of gastric mucosal tissues and analysis of gastric mucosal injury index in the mouse model treated with Mito-TEMPO or not. Scale bar, 100 µm. n=6 per group. * P<0.05 vs. the control group; # P<0.05 vs. the PHT group without Mito-TEMPO. (B) The morphology and H&E staining of GES-1 cell under normoxic and hypoxic conditions treated with Mito-TEMPO (or not) are shown. The relative cell viability analyzed by Cell Counting Kit-8 were also determined. Scale bar, 25 µm. n=6 per group. * P<0.05 vs. the normoxia group; # P<0.05 vs. the hypoxia group without Mito-TEMPO. ROS, reactive oxygen species; H&E, hematoxylin and eosin; PHT, portal hypertension; CTRL, control.

Article Snippet: To mitigate oxidative stress, mice received intraperitoneal Mito-TEMPO (10 mg/kg solubilized in PBS; cat. no. HY-112879; MedChemExpress) every other day.

Techniques: In Vivo, In Vitro, Staining, Control, Cell Counting

Journal: Nature Immunology

Article Title: Metabolic adaptations rewire CD4 + T cells in a subset-specific manner in human critical illness with and without sepsis

doi: 10.1038/s41590-025-02390-6

Figure Lengend Snippet: a , GSEA showing running enrichment scores for ‘ROS pathway’ from the Hallmark library for CI-Sep ( n = 19) versus NHC ( n = 9) CD4 + T N , T EM and T reg cells. Gene ranks were determined by DESeq2 analysis of pseudobulked scRNA-seq transcriptomes. b , Dot plot showing single-cell normalized, log-transformed mean expression of selected ROS and antioxidant genes in CD4 + T N , T EM and T reg cells from NHC ( n = 9) and CI-Sep ( n = 19). c , d Representative flow cytometry plots ( c ) and quantification of mitochondrial ROS ( d ) determined by MitoSOX Green staining in CD4 + T N , T EM and T reg cells isolated on day 2 post-ICU admission from NHC ( n = 9), CI-NS ( n = 18) and CI-Sep ( n = 19). MitoSOX hi denotes the cells in the gated region. Percentages are of the CD4 + T cell subset parent gate. MitoSOX MFI shows the per-cell intensity of MitoSOX staining across all cells in each CD4 + T cell subset. e , Glycolytic capacity by puromycin-incorporation assay in NHC ( n = 6), CI-NS ( n = 7) and CI-Sep ( n = 8) CD4 + T N , T EM and T reg cells from paired PBMC cultures treated with vehicle control or MitoTEMPO (10 μM) for 24 h. Displayed as percentage of the maximum puromycin incorporation without metabolic inhibitors. f , Percentage change in FOXP3 MFI among CD4 + FOXP3 + T reg cells (left) and percentage change in the frequency of TIGIT + cells among CD4 + FOXP3 + T reg cells (right) from PBMCs stimulated with CD3/CD28/CD2 antibodies for 24 h and low-dose oligomycin (10 nM) with or without MitoTEMPO relative to CD3/CD28/CD2 antibody-stimulated, oligomycin-untreated, without MitoTEMPO, PBMCs from NHC ( n = 8), CI-NS ( n = 6) and CI-Sep ( n = 10). g , Percentage change in the frequency of TNF + cells among CD4 + FOXP3 − T conv cells from stimulated, oligomycin-treated PBMCs with vehicle or MitoTEMPO relative to stimulated, untreated PBMCs isolated from NHC ( n = 8), CI-NS ( n = 6) and CI-Sep ( n = 10) donors and treated as in f . Statistical analysis was performed using permutation testing of the enrichment score (weighted Kolmogorov–Smirnov statistic) with Benjamini–Hochberg FDR correction for multiple testing ( a ); using the Kruskal–Wallis test with Dunn’s post hoc testing ( d ); and using paired sample testing with the two-sided Wilcoxon signed-rank test ( e – g ). Data for d–g are presented as mean ± s.e.m. Each data point represents an individual donor.

Article Snippet: Where indicated, cells were treated with low-dose oligomycin (Cayman Chemical, cat. no. 11342, 10 nM), GSK180 (MedChemExpress, cat. no. HY-112179, 10 μM) or MitoTEMPO (MedChemExpress, cat. no. HY-112879, 10 μM) for the full 24 h stimulation.

Techniques: Transformation Assay, Expressing, Flow Cytometry, Staining, Isolation, Control