Extended Data Fig. 1: Fumarate depletion by FH upregulation is essential for B-cell activation.

a, Schematic representation of TCA cycle. b and c, Mouse naive B cells were stimulated by 10 µg/ml (unless otherwise indicated) F(ab’)₂ fragments of antimouse IgM antibody (hereafter referred to as anti-IgM) for indicated times or left unstimulated (no stim). Cells were analyzed by western blot (b) and LC-MS for metabolites (c). Cit, Citrate; α-KG, alpha-ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; OAA, oxaloacetate. Stim stands for stimulation (n = 3 independent wells). d, Mouse naive B cells were treated with DMSO (Ctrl), indicated metabolites (1 mM) or 50 µM fumarate hydratase-IN-1 (FHi) for 1 hour prior to 24 hours of anti-IgM antibody treatment or left untreated (no stim). Surface expression of CD86⁺ and CD69⁺ on B cells were measured by FACS analysis (n = 3 independent wells). e, Mouse naive B cells were treated with DMSO, 50 µM FHi or 100 µM DMF as indicated for 1 hour before activation for indicated times. The percentage of CD86⁺ and CD69⁺ B cells was measured (n = 3 independent wells). f and g, Mouse naive B cells were treated with DMSO or increasing amounts of FHi or DMF for 1 hour prior to stimulation without (no stim) or with anti-IgM antibody for 24 hours. The MFI (f) and percentage (g) of CD86⁺ B cells and CD69⁺ B cells were measured (n = 3 independent wells). h and i, Mouse naive B cells expressing shFH or shCtrl were stimulated with anti-IgM antibody. The levels of metabolites (i), MFI and percentage of CD86⁺ and CD69⁺ B cells, and FH expression were measured (h). n = 3 independent wells. The data are normalized to the average of no stimulation samples(c) or the average of control samples (i). The data in c, g, h, and i are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. The data in d, e, and f are non-Gaussian distributed, and the differences were evaluated by using 2-tailed Mann-Whitney U test. Data are the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test or Mann-Whitney U test.

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