alexa fluor 647–conjugated cleaved caspase-3 antibody #9602s  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions

doi: 10.1038/s41467-024-51166-3

Figure Lengend Snippet: 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.

Article Snippet: Cells were stained 1:100 with an Alexa Fluor 647 anti-active caspase-3 antibody (Cat. 560626, BD), at 4 °C for 45 min. All flow cytometry experiments were conducted on FACSCelesta or LSRII flow cytometers (BD) and analyzed using FlowJo 10.8 Software.

Techniques: Mutagenesis, Staining, Flow Cytometry, Binding Assay, Ligation, Incubation, Expressing, Negative Staining, Imaging, Derivative Assay, Translocation Assay

Journal: Nature Communications

Article Title: TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions

doi: 10.1038/s41467-024-51166-3

Figure Lengend Snippet: 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. ). 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. ) 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.

Article Snippet: Cells were stained 1:100 with an Alexa Fluor 647 anti-active caspase-3 antibody (Cat. 560626, BD), at 4 °C for 45 min. All flow cytometry experiments were conducted on FACSCelesta or LSRII flow cytometers (BD) and analyzed using FlowJo 10.8 Software.

Techniques: Expressing, Imaging, Flow Cytometry, Staining, Fluorescence, Mutagenesis, Control, Two Tailed Test, Isolation

Journal: Nature Communications

Article Title: TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions

doi: 10.1038/s41467-024-51166-3

Figure Lengend Snippet: Antigenic stimulation induces nuclear translocation of TFEB. In the absence of T cell help, TFEB transcriptional activity leads to death-by-neglect through upregulation of pro-apoptotic proteins (e.g., BH3-only proteins, caspase-3). Concomitantly, TFEB arranges for a possible rescue by promotion of B cell migration to lymph follicles and GC entry (e.g. via regulation of CXCR4 and CXCR5) as well as induction of antigen presentation via MHC II. In the presence of T-lymphoid co-stimulation, CD40 rescue prevents TFEB-driven apoptosis by upregulation of Bcl-2 family proteins. CD40 signaling concurrently prolongs TFEB nuclear activity, further enhancing TFEB’s stimulatory influence on B cells, leading to further activation and maturation through DZ (re-)entry. This graphical summary was created with BioRender.com under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 international license.

Article Snippet: Cells were stained 1:100 with an Alexa Fluor 647 anti-active caspase-3 antibody (Cat. 560626, BD), at 4 °C for 45 min. All flow cytometry experiments were conducted on FACSCelesta or LSRII flow cytometers (BD) and analyzed using FlowJo 10.8 Software.

Techniques: Translocation Assay, Activity Assay, Migration, Activation Assay