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. 2024 Aug 14;15:6971. doi: 10.1038/s41467-024-51166-3

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — a–e Mouse splenocytes were left untreated or stimulated with anti-IgM/G for 60 min and analyzed for TFEB expression and subcellular distribution by imaging flow cytometry. a Staining strategy to distinguish germinal center and non-GC B cells from total splenocytes. GC B cells were gated as CD19⁺Fas⁺GL7⁺, whereas non-GC B cells were defined as CD19⁺Fas^-GL7^- (gating depicted in Supplementary Fig. 9a). b Representative multi-channel images of individual resting and BCR-stimulated germinal center and non-GC B cells. c Percentage of cells with translocated TFEB, as defined by TFEB/7-AAD similarity score ≥ 1. d Mean fluorescence intensity of TFEB. e Mean fluorescence intensity of TFEB within the nucleus. Data are depicted as mean ± SD of n = 4 littermates. Statistical significances were computed using RM two-way ANOVA, corrected for multiple testing via Tukey’s method. f–j Flow cytometric analyzes of GC subsets in B cell-conditional TFEB mutant mice (n = 13) and control littermates (n = 16). f GC B cells gated as B220⁺Fas⁺GL7⁺. were further distinguished in LZ and DZ GC B cells by their expression of CD86 and CXCR4. g Absolute number of splenic B220⁺ B cells. h Percentage of B220⁺Fas⁺GL7⁺ GC B cells among total B220⁺ B cells. i Percentage of LZ and DZ GC B cells among total GC B cells. j Ratio of LZ and DZ GC B cells. Data in (g–j) are depicted as mean ± SD. Statistical significances were computed using Šidák-corrected RM two-way ANOVA (i) or two-tailed Student’s t-test (g–j). k, l Splenocytes of TFEB-depleted and control mice were isolated and stimulated with anti-IgM/G and co-stimulated with CD40 for 18 h, as indicated. CD19⁺ B cells were further subgated into germinal center (CD19⁺Fas⁺GL7⁺) and non-GC B cells (CD19⁺Fas^-GL7^-; gating depicted in Supplementary Fig. 9b) and co-stained with anti-active caspase-3-AF647 and Zombie Green dye. k Selected histograms depicting active caspase-3 fluorescence intensity. l Proportions of active caspase-3-positive B cells among GC or non-GC B cells, depicted as mean ± SD of n = 3 independent experiments. Statistical significances were computed using RM two-way ANOVA and Fisher’s LSD test. Source data are provided as a Source Data file.