Fig 1: Transcriptome analysis of naive T and sAb-, sAb+LatA-, and iAb-stimulated naive T cells.a Coordinated changes in gene expression (Affymetrix Mouse Gene ST 2.0 microarray) in naive T (0 h) and LatA-, sAb-, sAb+LatA-, and iAb-stimulated T cells at 3 and 24 h. Genes on the heatmap were selected based on fold changes from naive T cells >1.5 and plotted with normalized gene expressions (z-score). b Principal component analysis (PCA) of the transcriptome. Trajectory was changed from naive T cells to sAb-, sAb+LatA-, and iAb-stimulated T cells. The changes were time-dependent, observing different clusters by time. c Selected heatmap of naive T and sAb- and iAb-activated cells at 3 h. Genes on the heatmap were selected as described in (a). Most of the genes in the selected group exhibited a reversed pattern of expression changes between naive cells and iAb-activated cells. d Enriched biological pathways (Gene Ontology) of differentially expressed genes. e Enriched metabolic pathways of T cells. NES of metabolic pathways (R fgsea package) were calculated from the fold changes of group comparisons. Pathways were selected based on enrichment test p values (<0.05). Bar graphs show the fold changes (log2) of representative genes in the FAS or FAO pathways. f Kinetic OCR response to palmitate. Naive CD3+ T cells stimulated for 3 h with iAb or sAb, washed, and OCR was measured in the presence of 500 μM palmitate. Results are representative of three independent experiments ± SD. p value was presented for iAb vs sAb. g Levels of FAO enzymes ACADM, ACADVL, and HADHA were measured by flow cytometry in sAb- and iAb-activated cells at 3 h or 24 h. Representative histogram and the mean MFI is shown. Results are representative of three independent experiments ± SEM. h A schematic model based on global metabolic gene profiling and the metabolic functional changes caused by molting or TCR internalization. Statistics was performed using one-way ANOVA with post hoc Tukey’s multiple comparisons test. FAS fatty acid synthesis, FAO fatty acid oxidation, GPI glycosylphosphatidylinositol, and OXPHOS oxidative phosphorylation, ACADM, medium-chain acyl-CoA dehydrogenase, ACADVL very long chain acyl-CoA dehydrogenase, HADHA trifunctional multi enzyme complex subunit alpha. Source data are provided as a Source Data file.
Fig 2: Impaired FAO and LD accumulation in elderly-derived monocytes(A–I) Blood monocytes were isolated from young and old mice. (A) Western blot analysis of p-AMPK and p-mTOR expression in each group. Representative blots of three independent experiments are shown. (B) ATP concentrations were tested using an ATP colorimetric/fluorometric assay and normalized by the protein concentration (n = 4 per group). (C) The OCR was tested in XF-96 assay medium and normalized by protein concentration. Continuous OCR values (pmoles/min/μg protein) are shown. Each repetition involved three mice per group, and three replicates were performed. (D) Western blot analysis of ACADVL, HADHA, and ACADM expression in each group. Representative blots of three independent experiments are shown. (E) FAO enzyme abundance in monocytes was detected by flow cytometry. Representative mean fluorescent intensity (MFIs) and the mean MFIs are shown (n = 4 per group). (F) Morphological analysis of young and old murine monocytes by electron microscopy. (G) LDs in monocytes were stained with BODIPY 493/503 dye and then imaged under a confocal microscope before 3D rendering using Imaris software (version 9.0). (H) The mean LD volume of 50 analyzed cells. (I) LD density in monocytes from young (red line) and old (blue line) mice were analyzed by flow cytometry.(J) Human blood monocytes were isolated by using two-step procedures, and LDs were identified in CD14+ cells by BODIPY 493/503 immunofluorescence staining.(K) BODIPY 493/503 MFI values in CD14+/CD16+ blood monocytes isolated from young and elderly human subjects.(L) Correlation analysis between age and monocytes, BODIPY MFI values. (n = 30).The resulting Spearman's correlation coefficient (r) and corresponding p value are reported. All data represent mean ± SD. ∗∗p < 0.01, ∗∗∗p < 0.001; t test (B, E, and H).
Supplier Page from Abcam for Fatty Acid Oxidation Assay Kit (flow cytometry)