Extended Data Fig. 8: HGPRT knockout effectively modulates nucleotide metabolism and significantly impacts macrophage gene expression.
a) Cell viability in wildtype and Hprt1 KO RAW 264.7 cells, either unstimulated or continually stimulated for 48 h. b) M + 1 labeled fraction of GTP in wildtype or Hprt1 KO (2 clones) RAW 264.7 cells, either unstimulated or continually stimulated for 48 h. Cells were labeled with γ-15N-glutamine for 24 h before analysis. c) Expression levels of Hprt1 and Aprt (encoding adenine phosphoribosyltransferase) in unstimulated wildtype or Hprt1 KO RAW 264.7 cells. Data are presented as fold change relative to wildtype unstimulated. d) Metabolomic changes in pentose phosphates, nucleotides, nucleosides, and nitrogenous bases in 48 h stimulated wildtype or Hprt1 KO (2 clones) RAW 264.7 cells. Metabolite abundances are relative to wildtype cells and displayed on a log2 scale as a heatmap. Each box represents the mean of n = 3 independent samples. e) Labeling pattern of M + 1 GTP in BMDMs over a timecourse of continual stimulation treated with or without 6-mercaptopurine. Cells were labeled with γ-15N-glutamine for 24 h before analysis. a–c, e) Mean ± SD (n = 3 independent samples). a, c, e) Statistical analysis was performed using unpaired two-tailed student’s t-test comparing WT to Hprt1 KO or untreated to 6-MP treated cells at each time point. ns indicates not significant (p > 0.01). b) 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. f–g) Volcano plot showing differentially expressed genes (up-regulated in red and down-regulated in green) in Hprt1 KO RAW 264.7 cells compared to wildtype cells in f) unstimulated state, and g) 48 h stimulated state.
