This study elucidates the essential role of pyrimidines in supporting mitochondrial pyruvate oxidation and the tricarboxylic acid (TCA) cycle. Quantitative assessment of metabolite changes in response to alterations in cellular pyrimidine levels was conducted using liquid chromatography-mass spectrometry. Concurrently, western blot analysis was employed to characterize the expression of metabolic enzymes potentially involved in this regulatory process. To enhance precision, metabolic activity measurements were conducted under both untreated and pyrimidine-depleted conditions using radioactivity-based assays, isotope tracers, and Seahorse analyzers. The comprehensive dataset includes experimental quantifications of metabolite abundance, accurate assessments of metabolic activity, and protein levels of metabolic and signaling enzymes.

1. Steady-State Metabolomics:

• Mass spectrometry extracted and identified metabolites from mammalian cells (HeLa, HEK293E) and yeast cells. Mouse liver samples, collected post-treatment, were ground to extract metabolites for mass spectrometry analysis.
• Reported metabolite abundance using peak areas as a measure.

2. Stable Isotope Tracing Experiments

• Cells or mice were labeled with a heavy isotope (e.g., 13C-glucose) to assess specific pathway metabolic activity using mass spectrometry.
• Fractional enrichment is reported, indicating newly synthesized metabolite abundance from the tracer.

3. Western Blot:

• Quantification of protein levels adhered to outlined purification procedures, with subsequent SDS-PAGE gel electrophoresis.
• Presentation of unprocessed western blot images.

4. Radioactivity-Based Assays:

• Cells labeled with radioactive materials (e.g., 14C-pyruvate or 14C-glucose), followed by extraction of RNA or lipids.
• Reported Count per Minute (CPM), reflecting ionization events per minute.

5. Oxygen consumption rate:

• Mammalian cells, treated as indicated in the manuscript, had their oxygen consumption rate measured using the Seahorse Analyzer instrument.