Journal: Nature metabolism

Article Title: Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle

doi: 10.1038/s42255-023-00735-9

Figure Lengend Snippet: ( A ) Heatmap of LC/MS-analyzed 122 polar metabolites from double thymidine block (DTB)-synchronized HeLa cells. Analytes are clustered and colored by metabolic pathway. Carbamoyl-aspartate, dihydroorotate, and orotate are highlighted in purple. The presented result is in logarithmic ratio (log2 fold change relative to DTB) of the mean of biological triplicate (n = 3, biological replicates). ( B ) De novo pyrimidine biosynthesis pathway. Carbons are depicted in a circle; nitrogens, square; ribose, pentagon; phosphate groups, a P in a circle; Amide- 15 N of glutamine, red. ( C ) Relative changes of intracellular amide- 15 N glutamine-derived 15 N-dihydroorotate concentration after DTB release (left) or post-nocodazole (NOC) release (right). 1 hour amide- 15 N glutamine labeling. 15 N-labeled dihydroorotate, pink, unlabeled dihydroorotate, black. Data are presented as mean (n = 3, biological replicates) +/− SD (standard deviation). Two-tailed Student t-test statistical analyses applied. ( D ) Genomic incorporation of EdU. Uridine (100 μg/mL) PALA (300 μM, 24 h) as indicated. ( E ) Relative intracellular dihydroorotate concentration in wild-type HeLa, sgCAD, and wild-type CAD-reconstituted sgCAD cells. Data are presented as mean +/− SD (n = 3, biological replicates). Two-tailed Student t-test statistical analyses applied. Two-tailed Student’s t-test statistical analyses applied. ( F ) Cell proliferation of sgCAD cells upon supplementation of 500 μM of indicated molecules. 4 days of supplementation duration. Data are presented as mean +/− SD (n = 3, technical replicates, 3 independent experiments). Two-tailed Student’s t-test statistical analyses applied. ( G ) Cell proliferation of wild-type HeLa, sgCAD, and wild-type CAD-reconstituted sgCAD cells upon uridine supplementation, as indicated. Data are presented as mean +/− SD (n = 3, technical replicates, 2 independent experiments). Two-tailed Student’s t-test statistical analyses applied. Compared to the cells with 0 μg/mL. ( H ) Immunoblotting of lysates from sgCAD at indicated time after uridine withdrawal or uridine supplementation (100 μg/mL). P-values denoted as * (* for p-value < 0.1; **, < 0.01; ***, < 0.001; ****, < 0.0001) or ns (not significant).

Article Snippet: The dendrogram was paired with a heatmap (plotly.graph_objects) of log 2 fold change of metabolites.

Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Blocking Assay, Derivative Assay, Concentration Assay, Labeling, Standard Deviation, Two Tailed Test, Western Blot