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. 2024 Jan 23;25(3):552–561. doi: 10.1038/s41590-023-01738-0

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© The Author(s) 2024

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. 3 — a, Immunofluorescence staining of LDHB and COX IV in CD8⁺ T_eff cells treated with LA and/or LC. Scale bar, 5 μm (left). Pearson’s correlation coefficient between LDHB and COX IV (right). n = 6 cells examined over three independent experiments. b, Immunofluorescence staining of TFEB in CD8⁺ T_eff cells as in a. Scale bar, 10 μm (left). Pearson’s correlation coefficient between TFEB and DAPI (right). n = 20 cells examined over three independent experiments. c, ChIP–qPCR analysis of TFEB enrichment around the promoter of LDHB in CD8⁺ T_eff cells. LDHB primer 1 and 2 were used. n = 5. d, Relative luciferase activity of HEK-293T cells transfected with Tfeb-overexpressing (oeTfeb) or LDHB-promoter (LDHB-pro) plasmids. n = 6. e, Immunoblots of LDHB in Tfeb^fl/fl or Tfeb^fl/fl Lck^cre mice spleen-derived CD8⁺ T_eff cells. f, Relative abundance of mitochondrial labeled lactate, citrate and malate derived from [¹³C]lactate in CD8⁺ T_eff cells of Tfeb^fl/fl or Tfeb^fl/fl Lck^cre mice. n = 7 in M + 2 citrate Tfeb^fl/fl CD8⁺ T group, n = 8 in other groups. g, Fluo-4 mean fluorescence intensity (MFI) of cytosolic calcium release in CD8⁺ T_eff cells pretreated with CGP37157 (CGP, 10 μM) or ryanodine (Ryn, 100 μM) for 1 h then treated with LA and/or LC. h, Fluo-4 MFI of cytosolic calcium release in CD8⁺ T_eff cells pre-transduced with shMcoln2 then treated with LA and/or LC. i,j, Immunoblots of TFEB (i) and LDHB (j) in CD8⁺ T_eff cells as in h. k, Lysosomal pH value of CD8⁺ T_eff cells was detected. n = 6. l, Schematic of experimental design for deuterium detection. Created with BioRender.com. m, Lysosomal ²H concentration, determined by isotope-MS, of CD8⁺ T_eff cells with or without deuterated ²H-acetic acid (DAC, 10 mM) or ²H-sulfuric acid (D₂SO₄, 5 mM) for 3 h. n = 4. n, Lysosomal Li⁺ concentration in CD8⁺ T_eff cells treated with LC/LiCl and/or bafilomycin A1 (Baf A1, 1 nM) was detected. n = 6 (left) and n = 5 (right). Data are the mean ± s.e.m. n = biological replicates unless stated otherwise. P values were calculated using one-way ANOVA for Dunnett’s multiple-comparisons test (a–d, f, k, and m) and Kruskal–Wallis test (n).

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