Journal: Nature Immunology
Article Title: Dynamic mitochondrial transcription and translation in B cells control germinal center entry and lymphomagenesis
doi: 10.1038/s41590-023-01484-3
Figure Lengend Snippet: a , Representative Airyscan IHC images of spleen sections depicting GCs. Quantification of the dark zone area normalized to the GC area in SRBC-immunized Aicda-WT ( n = 41 GCs) and Aicda-Tfam ( n = 17 GCs) mice. Image analyses were performed including all GCs identified on two sections from n = 2 mice of each genotype. Scale bar, 100 μm. Representative of four independent experiments. b , Representative flow cytometry plots of the dark zone, gray zone and light zone subpopulations of tdTomato + CD38 − GL-7 + GC B cells. Quantification of the dark zone to light zone ratio from NP-CGG-immunized Aicda-WT ( n = 8) and Aicda-Tfam ( n = 9) mice is shown. Representative of four independent experiments. c , 3D Airyscan confocal images of magnetic bead-sorted and F-actin phalloidin + 4,6-diamidino-2-phenylindole (DAPI)-stained GC B cells from Aicda-WT and Aicda-Tfam mice. Scale bar, 6 μm. Representative of two independent experiments. d , Representative flow cytometry histogram of F-actin phalloidin fluorescence of tdTomato + GL-7 + GC B cells and tdTomato − IgD + naive B cells from immunized Aicda-WT ( n = 6) and Aicda-Tfam ( n = 8) mice. Quantification of F-actin phalloidin gMFI in GC B cells normalized to naive B cells from the same host is shown. Data were pooled from three independent experiments. e , Quantification of chemotaxis to CXCL12 and CXCL13 in GC B cells from Aicda-WT ( n = 3) and Aicda-Tfam ( n = 5) mice. Data are representative of four independent experiments. f , Representative Fluo-4 AM geometric mean signal intensity kinetics (moving average) of B-WT ( n = 4) and B-Tfam ( n = 4 mice) B220 + B cells stimulated with CXCL12 for 120 s. Quantification of the area under the curve (AUC) between 60 and 90 s (corresponding to the peak response) is shown. g , Representative Fluo-4 AM gMFI signal kinetics (moving average) of B-WT ( n = 4) and B-Tfam ( n = 4 mice) B220 + B cells stimulated with anti-IgM for 300 s. Quantification of the AUC between 60 and 300 s is shown. In f and g , experiments were run as technical duplicates in four independent replicate experiments consisting of one B-Tfam and one WT mouse in each. The data points from the B-Tfam mice were normalized to the WT data run in the same batch. h , Representative flow cytometry histogram of MCU fluorescence in B220 + IgD + B cells from unimmunized B-Tfam and B-WT mice. Quantification of MCU gMFI in IgD + B cells from B-Tfam and B-WT mice ( n = 3). Data are representative of three independent experiments. i , Representative flow cytometry histogram of mtROS-Deep Red fluorescence in IgD + B cells from B-WT ( n = 4) and B-Tfam ( n = 5) mice. Data are representative of two independent experiments. j , Quantification of chemotaxis to CXCL12 in B cells from unimmunized B-Tfam ( n = 3) and B-WT ( n = 5) mice in the presence of mitoTEMPO or Ru265. Data are representative of two independent experiments and were pooled after batch correction. Statistical significance was calculated using an unpaired two-tailed t -test ( b , d , h , i ), a two-tailed Mann–Whitney U -test ( a , f , g ) or a two-way ANOVA with Šidák’s correction ( e ), with batch as a covariate ( j ). Data are presented as the mean ± s.e.m.
Article Snippet: A total of 2 × 10 5 purified total B cells from B-Tfam and B-WT mice were placed in a 6.5-mm transwell chamber with 5-μm pore size and incubated for 5 h in the presence or absence of murine CXCL12 (100 ng ml −1 , BioLegend) with or without mitoTempo (100 μM, Merck) and Ru265 (30 μM, Merck).
Techniques: Flow Cytometry, Staining, Fluorescence, Chemotaxis Assay, Two Tailed Test, MANN-WHITNEY
