Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis
Data files
Mar 13, 2024 version files 11.63 MB
-
README.md
1.93 KB
-
Source_data_Fig_1.xlsx
42.01 KB
-
Source_data_Fig_2.xlsx
36.97 KB
-
Source_data_Fig_3.xlsx
30.99 KB
-
Source_data_Fig_4.xlsx
35.73 KB
-
Source_data_Fig_5.xlsx
20.76 KB
-
Source_data_Fig_S1.xlsx
21.34 KB
-
Source_data_Fig_S10.xlsx
13.31 KB
-
Source_data_Fig_S11.xlsx
23.36 KB
-
Source_data_Fig_S12.xlsx
13.60 KB
-
Source_data_Fig_S3.xlsx
34.77 KB
-
Source_data_Fig_S4.xlsx
37.75 KB
-
Source_data_Fig_S5.xlsx
20.76 KB
-
Source_data_Fig_S6.xlsx
20.55 KB
-
Source_data_Fig_S7.xlsx
15.14 KB
-
Source_data_Fig_S8.xlsx
26.17 KB
-
Source_data_Fig_S9.xlsx
19.92 KB
-
Uprocessed_blots_and_images-adh2771.pdf
11.21 MB
Abstract
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.
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
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.