Extended Data Fig. 3: Metabolic reprogramming in LPS-stimulated BMDMs.
a,b, Oxygen consumption rate (OCR) measured by Seahorse XF mito stress test in nonstimulated (NS) BMDMs and BMDMs stimulated with LPS for 3 h, 6 h or 24 h (n = 3). Data are mean +/- S.E.M. of biological replicates. c, Extracellular acidification rate (ECAR) in BMDMs stimulated with LPS for 3 h, 6 h or 24 h or NS measured by Seahorse (n = 3). Data are mean +/- S.E.M. of biological replicates. d, Extracellular lactate measured by mass spectrometry in BMDMs stimulated with LPS for 3–24 h or NS (n = 6) (12 h LPS, P = 3×10-5). Data are mean +/- S.E.M. of biological replicates. e, Western blot of hexokinase I, GAPDH, PDH, PKM2, PKM1/2 and tubulin in BMDMs treated with LPS for 3–24 h or NS (n = 3). Tubulin is a representative blot. Blots show three biological replicates for each condition. f-h, Quantification of protein determined by western blotting, normalized to NS and tubulin loading control +/- S.E.M. of biological replicates (n = 3). PKM2 was also normalized to PKM1/2. i,j, Metabolites measured by mass spectrometry in BMDMs stimulated with LPS for 6 h or 24 h or nonstimulated (NS) (n = 3). Data depicts z-score of biological replicates. k,l, Western blot and quantification of IRG1 normalized to vinculin loading control in BMDMs treated with LPS for 3–24 h or NS (n = 3). Blots show three biological replicates for each condition and data are mean normalized to NS and vinculin loading control. m,n, Succinate and itaconate measured by mass spectrometry in BMDMs stimulated with LPS for 6 h or NS, non-treated or treated with 10 μM BPTES for 6 h (n = 3) (6 h LPS Control vs BPTES, P = 1×10-6). Data are mean +/- S.E.M. of biological replicates. P values displayed above graphs calculated using two-tailed Student’s t-test for paired comparisons or one-way ANOVA for multiple comparisons.
