Fig. 6: Glutamine metabolism feeds CD40-induced lactate production.
a, mRNA expression of pro-inflammatory marker genes in BMDMs stimulated with Ctrl or FGK45 mAb under glucose (Glc) or glutamine (Glu) -replete or -deplete conditions. b, Representative immunoblots of indicated proteins in Cas9-expressing BMDMs harboring indicated sgRNAs upon FGK45 mAb treatment under glucose-deplete media. c,d, The change of OCR (c, left), the average of basal OCR (c, right) and SRC (d) in BMDMs stimulated with either Ctrl or FGK45 mAb under glucose (Glc) or glutamine (Glu) -replete or -deplete conditions. e,f, The ratios of NAD+ to NADH (e), the change of ECAR (f, left) and the average of basal ECAR (f, right) in BMDMs stimulated with either Ctrl or FGK45 mAb in indicated conditions. g,h, Tumor growth (g) and tumor weight (h) in tumor-bearing mice transplanted with either indicated bone marrows under indicated treatments. i, Relative fold change in mRNA expression of pro-inflammatory marker genes in TAMs isolated from indicated tumor-bearing mice. j, Extracellular lactate production from Cas9-expressing BMDMs harboring indicated sgRNAs upon FGK45 treatment. k,l, qPCR analysis of mRNA expression of pro-inflammatory marker genes (k) and extracellular lactate production (l) in BMDMs harboring indicated sgRNAs upon stimulation. m, qPCR analysis of mRNA expression of pro-inflammatory marker genes in BMDMs stimulated in the absence or presence of malic enzyme inhibitor (ME1). n, mRNA expression of pro-inflammatory marker genes in Cas9-expressing BMDMs harboring indicated sgRNAs in the absence or presence of sodium pyruvate (Pyr). o,p, Representative immunoblots of indicated proteins (o) and mRNA expression of pro-inflammatory marker genes (p) in Cas9-expressing BMDMs harboring indicated sgRNAs stimulated with or without FGK45 mAb with or without NR. Data are representative of three independent experiments with n = 3 per group in a, b and j–p and n = 4 per group in c, d and f. g,i,o,p, Representative data or pooled data from two individual experiments with n = 4 mice per group in g; n = 4 for Ctrl-PBS, n = 4 for Ctrl-FGK45, n = 8 for GLS gRNA-PBS and n = 11 for GLS gRNA-FGK45 in h and n = 8 (i). All data are the mean ± s.d. and analyzed by a two-tailed Student’s t-test.
