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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. 7 — a Parental and TFEB-mutant WEHI-231 B cells were left untreated or BCR-stimulated for the indicated periods. Flow cytometric co-staining with Annexin V-BV421 and 7-AAD was used to detect early (Annexin V⁺/7-AAD^-) and late apoptosis (Annexin V⁺/7-AAD⁺). b Flow cytometry analysis of active caspase-3 in WEHI-231 cells treated as described above. Data in (a, b) are depicted as mean percentage±SD of gated cells from n = 3 independent experiments. Significances were computed using Dunnett-corrected two-way ANOVA. c BH3 profiling of TFEB-depleted and parental WEHI-231 cells using the indicated BH3-agonistic peptides. Binding specificities towards Bcl-2 family proteins are indicated in the corresponding matrix. Cytochrome c release was monitored for resting cells or after 6 h of BCR ligation by flow cytometry. BCR-induced sensitization is presented as mean ± SD percent difference (Δ%) between n = 4 technical replicates of stimulated versus unstimulated cells. Depicted data are representative of n = 3 biological replicates. Statistical significances were calculated using Tukey-corrected one-way ANOVA. d WEHI-231 cells were incubated with the indicated combinations of anti-BCR and anti-CD40 antibodies for 6 h. Intracellular Bcl-xL expression was assessed by flow cytometry and is depicted as MFI, normalized to unstimulated parental cells. e Parental WEHI-231 and TFEB KO #A-59 cells were left untreated or stimulated, as indicated. Medium was changed on day 3, and cells were left untreated or were rescued with anti-CD40 until day 6. On day 3 and day 6, cells were incubated with Annexin V/7-AAD to access viability, as defined by a double negative staining. f WEHI-231 cells were BCR-stimulated for the indicated time periods in the presence or absence of CD40 ligation. Imaging flow cytometry-derived representative images and nuclear translocation of TFEB as percentages of cells with a TFEB/7-AAD similarity score ≥ 1. g, h Primary peripheral human CD19⁺ B cells were left untreated or BCR-stimulated for 24 h with or without CD40 ligation. Nuclear translocation (g) and expression (h) of TFEB was measured by imaging flow cytometry. Data shown in (d–h) is presented as mean ± SD of n = 3 (d) or n = 4 (e–h) independent experiments. Statistical significances were calculated using Tukey-corrected one-way ANOVA. Source data are provided as a Source Data file.