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. 2023 May 25;25(6):877–891. doi: 10.1038/s41556-023-01152-6

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Sorted mouse pulmonary aaMAIT cell subsets were analysed for mitochondrial content and function at >40 days post BRD509 vaccination. a, Representative images. m, mitochondria; ld, lipid droplet; g, granule. Scale bar, 1 μm. b,c, Quantification of mitochondrial number and morphology (b) and cytotoxic granule count (c) from transmission electron micrograph sections of aaMAIT cell subsets. ^n.s.P = 0.504661. **P = 0.003697, **P = 0.003851; unpaired two-tailed t-tests. In b, n = 22, 22, 21, 54, 22 and 54 cells from five pooled mice per each group. In c, n = 24 and 29 cells per group. d–f, Representative images (d) and quantification (e and f) of Bodipy493/503-positive lipid droplets (green) and mitochondrial Tom20 protein (purple) from z-stack composite airy-scan confocal micrographs of sorted aaMAIT cell subsets. Hoechst 33342 demarcates nucleus (blue). Scale bar, 5 μm; **P = 0.00130, ^n.s.P = 0.9; Mann–Whitney, two-tailed. n = 25, 20, 25 and 20 cells per group, pooled from five mice per group over two independent experiments. g–i, Representative images (g) and quantification (h and i) of mitochondrial ΔΨ_m by MTR DR FM intensity. Hoechst 33342 demarcates nucleus (blue, in bottom row combined channel images). Scale bar, 5 μm. Quantification represents the area (h) and count (i) of mitochondria that have high or low ΔΨ_m signal, respectively. n = 27 and 20 cells per group, pooled from five mice per group over two independent experiments. ***P = 0.0005 (h), ***P = 0.0004, *P = 0.047 (i); all Mann–Whitney, two-tailed. j, OCR kinetics (left) in response to injection of Oligomycin (Oligo), FCCP, Rotenone (Rot) and Antimycin A (AA) measured by Seahorse Bioanalyzer in sorted subsets of pulmonary aaMAIT cell subsets 40 days after vaccination with BRD509. OCR (middle) and basal ECAR (right) were measured without drug treatments or stimulation. **P = 0.0072, ^n.s.P = 0.84; unpaired, two-tailed t-tests. n = 8, 7, 6 and 4 independent samples per group, pooled from ten mice per group from one of two experiments. k, Quantification of mitochondrial ΔΨ_m by MTR DR FM and uptake of Bodipy-FL C16 FA in MAIT cell subsets isolated from lungs 40 days post vaccination with BRD509 and cultured as pulmonary cell suspensions for 18 h with or without re-activation with antigen (5-OP-RU), cytokines (IL-2/-12/-18) or the combination. n = 6, 3 and 3 independent samples per group from one experiment. All data displayed as mean ± s.e.m. Source numerical data are available in source data. n.s., not significant.

Source data