Extended Data Fig. 7: Rspondin3 mediated increase in mitochondrial respiration through glutamine metabolism.
a, Basal extracellular acidification rate (ECAR) measured in macrophages stimulated with Rspondin3, LPS alone or in combination for 24h with complete DMEM medium containing 10 mM glucose, 2 mM glutamine, 2 mM sodium pyruvate as substrate; Data are representative of three independent experiments, with 10-12 samples per group. Graphs show the mean ± s.d, with each dot representing an individual sample per time point. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism, P values are ***P=0.0003, ****P<0.0001. b, Basal ECAR measured in macrophages stimulated with Rspondin3, LPS alone or in combination with only 2 mM glutamine as substrate; Data are representative of three independent experiments, with 10-12 samples per group. Graphs show the mean ± s.d, with each dot representing an individual sample per time point. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism, P values are ****P<0.0001, ****P<0.0001. c, Basal ECAR measured in BMDMs stimulated with Rspondin3, LPS alone or in combination with only 10 nM free fatty acid as palmitate bound to BSA added as substrate; Data are representative of three independent experiments, with 10-12 samples per group. Graphs show the mean ± s.d, with each dot representing an individual sample per time point. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism, P values are ns P=0.9681, ns P=0.3389. d, Basal ECAR measured in BMDMs stimulated with Rspondin3, LPS alone or in combination with only10 mM glucose as substrate; Data are representative of three independent experiments, with 10-12 samples per group. Graphs show the mean ± s.d, with each dot representing an individual sample per time point. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism, P values are ns P=0.9694, ns P=0.9000. e, Basal ECAR measured in WT, Lgr4Mφ-/- and Ctnnb1Mφ-/- macrophages stimulated with or without Rspondin3 with only 2 mM glutamine as substrate; Data are representative of three independent experiments, graphs show the mean ± s.d, with each dot representing an individual sample per time point. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism, P values are **P=0.0041, ns P>0.9999, ns P>0.9999. f, Glutaminase activity measured in WT, Lgr4Mφ-/- and Ctnnb1Mφ-/- BMDMs under basal, or stimulated with Rspondin3, LPS alone or in combination. Data are representative of three independent experiments (mean ± sd); Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism. P values (left to right) are ****P<0.0001, ****P<0.0001, ns P=0.9995, ns P>0.9999, ns P=0.9881, ns P>0.9999. g, Relative gene expression of glutaminase genes – Gls and Gls2 in WT, Lgr4Mφ-/- and Ctnnb1Mφ-/- BMDMs with or without Rspondin3 stimulation measured by qPCR; Data are representative of three independent experiments, n=3 samples from three individual mice per group (mean ± sd), statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism. P values are ns P>0.9999, ****P<0.0001.
