Extended Data Fig. 6: Glutamine metabolism supports CD40-mediated macrophage polarization.
(a) qPCR analysis of mRNA expression of proinflammatory marker genes in Cas9-expressing BMDMs harboring control sgRNA (Ctrl) and GLS gRNAs (GLS sgRNA-1 and -2) with or without FGK45 mAb stimulation. (b–c) OCR measurement (b; left), the average of basal OCR (b; right), and NAD+-TO-NADH ratios (c) in Cas9-expressing BMDMs harboring control sgRNA (Ctrl) and GLS gRNAs (GLS sgRNA-1 and -2) stimulated with or without FGK45 mAb under glucose-deplete media. (d, e) ECAR measurement (d; left), the average of basal ECAR (d; right), and extracellular lactate production (e) in Cas9-expressing BMDMs harboring control sgRNA (Ctrl) and GLS gRNAs (GLS sgRNA-1 and -2) stimulated with or without FGK45 mAb under glucose-deplete media. (f) qPCR analysis of mRNA expression of proinflammatory marker genes in BMDMs stimulated with or without FGK45 mAb in the absence or presence of PEPCK inhibitor (iPEPCK) under glucose-deplete media. (g) Representative immunoblots of MDH1 and β-actin in Cas9-expressing BMDMs harboring control sgRNA (Ctrl) and MDH1 gRNAs (MDH1 sgRNA-1 and -2). (h) qPCR analysis of mRNA expression of proinflammatory marker genes in Cas9-expressing BMDMs harboring control sgRNA (Ctrl) and MDH1 gRNAs (MDH1 sgRNA-1 and -2) stimulated with or without FGK45 mAb. (a–h) Representative data from three individual experiments (n = 3 per group in a, e, f, h; n = 4 per group in b-d). All data are presented as mean ± SD and analyzed by a two-tailed Student’s t-test.
