Extended Data Fig. 7: NO is a key regulator mediating the rewiring in nucleotide metabolism that is specific to classical activation.
a) Arginine uptake, as measured by the changes in arginine level in spent media compared to fresh media after a 22 h incubation period, by BMDMs over a timecourse of continual and acute stimulation. Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett’s test comparing all groups to unstimulated cells (0 h). ns indicates not significant (p > 0.05). b) p-mTOR, mTOR, p-p70S6K, p70S6K, p-4E-BP1, and 4E-BP1 protein levels in unstimulated Nos2−/− BMDMs over a timecourse of 200 µM DETA-NONOate treatment. c) p-p70S6K, p70S6K, p-4E-BP1, and 4E-BP1 protein levels in wildtype or Nos2−/− BMDMs that are unstimulated or continually stimulated with LPS + IFNγ, with or without treatment of 200 µM DETA-NONOate for 48 h. d) Schematic of differential arginine metabolism by classically activated and alternatively activated macrophages. e) Citrulline and ornithine abundance in RAW 264.7 cells over a timecourse of continual LPS + IFNγ (red) or IL-4 + IL-13 (blue) treatment. Data are presented relative to abundance in unstimulated cells (0 h). Statistical analysis was performed with unpaired two-tailed student’s t-test comparing stimulated groups at each time point. ns indicates not significant (p > 0.01). a, e) Mean ± SD (n = 3 independent samples). f) Schematic of the in vivo stimulation experiment. g–h) The fold changes of (g) dTTP and (h) IMP in CD11b+F4/80+ cells harvested from wildtype or Nos2−/− mice treated with LPS + IFNγ. Data are presented relative to abundance in CD11b+F4/80+ cells harvested from PBS injection controls from each trial. Mean ± SEM (n = 9 independent mice for PBS injection controls. n = 5 for LPS + IFNγ samples, with each replicate consisting of cells pooled together from two stimulated mice). g–h) Statistical analysis was performed with paired two-tailed student’s t-test comparing stimulated cells. i–j) The fold changes of (i) dTTP and (j) IMP in wildtype or Nos2−/− peritoneal macrophages after 48 h continual stimulation with LPS + IFNγ after isolation (ex vivo), compared to unstimulated control. Mean ± SD (n = 3 independent mice). Statistical analysis was performed with unpaired two-tailed student’s t-test comparing stimulated cells.
