Extended Data Fig. 3: DHODH is indispensable for the synthesis of orotate.
From: De novo pyrimidine biosynthetic complexes support cancer cell proliferation and ferroptosis defence
a, A schematic to show the metabolism of isotope-labeled glutamine via the reductive and oxidative pathways. b,c, Fractional labeling (b) and relative levels (c) of carbamoyl aspartate, dihydroorotate, orotate and UMP in WT and DHODH−/− Hela cells that were supplemented with 400 μM uridine and cultured with 1 mM 13C5-glutamine for 8 h before harvest. d, In vitro pull-down analysis of the interaction between GST-tagged proteins and His-VDAC3 purified from E. coli with GST protein as a negative control. e, HEK293T cells expressing Flag-tagged truncations of CAD were subjected to IP analysis using anti-Flag M2 beads, followed by immunoblotting. f, HEK293T cells expressing Flag-tagged truncations of DHODH were subjected to IP analysis using anti-Flag M2 beads, followed by immunoblotting. N-terminal region, 1-99aa; C-terminal region, 100-395aa. g, HeLa cells were homogenized and fractionated. The cytosolic and intact mitochondrial fractions were analyzed by immunoblotting. (b,c) Values are shown as the mean ± SD, n = 3 biologically independent samples. P values were obtained using unpaired two-tailed Student’s t-test. (d-g) Data are verified in three replicates with similar results.
