mitotempo  (MedChemExpress)

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