Extended Data Fig. 4: One-carbon metabolism supplements do not rescue the stimulation-induced inhibition of nucleotide de novo synthesis.
a) Transcriptional changes of detected genes in one carbon metabolism and serine de novo synthesis in BMDMs over a timecourse of acute stimulation. Relative expression of each gene is presented as relative to unstimulated cells (0 h) on a log2 scale as a heatmap, with saturating color representing 10-fold change (or 3.32 on log2 scale). Each box represents the mean of n = 3 independent samples. b–e) Labeled fraction of (b) M + 1 labeled AICAR, (c) M + 2 labeled IMP, (d) M + 2 labeled ATP, and (e) M + 2 labeled GTP in unstimulated or stimulated BMDMs supplemented with 1 mM 13C-formate for 24 h. f) Relative total intracellular dTTP abundance in unstimulated or stimulated BMDMs supplemented with 1 mM 13C-formate for 24 h. Data are presented relative to abundance in unstimulated cells. g–i) Labeled fraction of (g) IMP (M + 2) and (h) dTTP (M + 1), and (i) relative intracellular dTTP abundance in RAW 264.7 cells that are unstimulated, stimulated, or unstimulated and treated with 200 nM methotrexate (MTX), labeled with γ-15N-glutamine for 24 h. Cells were additionally supplemented with or without 1 µM folinate throughout the timecourse. b–i) Stimulated cells are continually stimulated for 48 h. Mean ± SD (n = 3 independent samples). b–f) Statistical analysis was performed with unpaired two-tailed student’s t-test comparing unstimulated to stimulated cells. g–i) Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test. Bars with different lower-case letters (a, b, c, or d) indicate a statistically significant difference with p < 0.05.
