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. 2023 May 11;4(5):648–664. doi: 10.1038/s43018-023-00556-5

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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. 5 — a, Oxygen consumption rate (OCR) was measured in GFP⁺ P3 cells sorted after transfer with a high or low amount of mitoDsRed⁺ mitochondria from donor mitoDsRed⁺ astrocytes and was compared with that in sorted GFP⁺mitoDsRed^– P3 cells from the same co-culture. Following readings of basal respiration, the stepwise addition of 3 mM oligomycin (Oligo) to measure leak respiration, 1.5 mM CCCP to quantify maximal and reserve capacity and 1 mM rotenone (ROT) followed by 1 mM antimycin A (AMA) to measure non-mitochondrial respiration was performed; CTR, control. b, Basal oxygen consumption gradually increased with cumulative mitochondrial content in GFP⁺ P3 cells. c, Maximal respiratory capacity gradually increased with cumulative mitochondria content in GFP⁺ P3 cells. d, Energy map indicating that GFP⁺ P3 cells with a higher degree of mitochondria transfer from astrocytes have a more aerobic and energetic phenotype than GFP⁺ P3 cells with less and no mitochondria transfer. Data in a–d are from a representative experiment from a total of three independent experiments. Data shown as mean ± s.e.m.; n = 18 (control), 22 (mitoDsRed low) and 9 (mitoDsRed high) technical replicates; statistical comparison of technical replicates is not shown. ECAR, extracellular acidification rate; mpH, milli pH. e, Seven distinct human-derived GSCs were cocultured for 4 d with immortalized mito-mCherry⁺ human astrocytes and stained with antibodies recognizing key metabolic proteins for downstream flow cytometry quantification. Protein expression was compared between mito-mCherry⁺ and mito-mCherry⁻ cells by mixed-effects model analysis. The dotted line represents the statistical significance threshold (false-discovery rate (FDR) < 0.05). f, ATP levels in sorted mitoDsRed⁺ versus mitoDsRed^– cells from distinct human-derived GSCs, measured with the CellTiter-Glo luminescence assay; n = 3 independent experiments; *P = 0.04. Data were analyzed by two-tailed ratio paired t-test; RLU, relative light units. g, ATP levels in sorted mito-mCherry⁺ versus mito-mCherry⁻ cells from three distinct human-derived GSC lines, assessed by CellTiter-Glo luminescence assay; n = 3 independent experiments; **P = 0.003. Data were analyzed by two-tailed t-test.

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