DOI: 10.5061/dryad.rxwdbrvfq
title: Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis

description: 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.
description: <strong>1. Steady-State
 Metabolomics:</strong> 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. <strong>2.
 Stable Isotope Tracing Experiments</strong> 
 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. <strong>3. Western
 Blot:</strong> Quantification of
 protein levels adhered to outlined purification procedures, with
 subsequent SDS-PAGE gel electrophoresis. Presentation
 of unprocessed western blot images. 
 <strong>4. Radioactivity-Based
 Assays:</strong> 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.
 <strong>5. </strong><strong>Oxygen
 consumption rate:</strong> Mammalian
 cells, treated as indicated in the manuscript, had their oxygen
 consumption rate measured using the Seahorse Analyzer
 instrument.
description: # Pyrimidines maintain mitochondrial pyruvate oxidation to support *de
 novo* lipogenesis
 [https://doi.org/10.5061/dryad.rxwdbrvfq](https://doi.org/10.5061/dryad.rxwdbrvfq) ## Description of the data and file structure The data in this study were derived from various sources, including various mammalian cell lines, mouse tissues, and a yeast strain (*Saccharomyces cerevisiae*), all comprehensively detailed in the main text and the supplemental materials and methods document. Our assessment of protein levels and metabolic activity was conducted through biochemical methods such as immunoblotting and mass spectrometry (metabolomics) (See methods). To quantify the levels of proteins of interest, we adhered to the purification procedures as outlined in the manuscript, subsequently running them on SDS-PAGE gels. These gels were then transferred onto nitrocellulose membranes and subjected to hybridization with specific antibodies for chemiluminescent detection. In the case of metabolite extraction, cultured cells or segments of mouse livers were either scraped or ground into the indicated solutions specified in the supplemental methods. The resulting metabolite suspensions were then prepared for mass spectrometry analysis. The files are systematically organized to align with the sequence of data presentation in the manuscript. Each image file is available in PDF format and contains the original, unprocessed images of western blots, cells, and figures, encompassing both those featured in the main text and supplementary materials. Additionally, the numerical data points for each graph in both the main and supplementary figures have been meticulously cataloged in separate Excel files, corresponding to the respective figure panels for ease of reference. Missing values or values below the detection threshold are denoted as NA or left empty. ## Sharing/Access information NA ## Code/Software NA