Extended Data Fig. 4: Involvement of VDAC3 in the pyrimidine biosynthetic complex.
From: De novo pyrimidine biosynthetic complexes support cancer cell proliferation and ferroptosis defence
a, Fractional labeling of carbamoyl aspartate, dihydroorotate, orotate and UMP in pooled VDAC3 knockout and control HeLa cells cultured in medium containing 1 mM 13C5-glutamine for 8 h. b, Pooled VDAC3 knockout and control HeLa cells were cultured in various concentrations of uridine. Cell viability was measured after 3 days of treatment. The results were normalized to those of cells infected with control sgRNA without uridine supplementation. The expression of VDAC3 was detected by immunoblotting. c, d, Proliferation of control and CAD silencing (c) or control and DHODH silencing (d) HeLa cells cultured in medium containing uridine (200 μM) or vehicle. The expression of CAD or DHODH was detected by immunoblotting. e, Cell viability of control and GOT1 silencing HeLa cells cultured with or without uridine (400 μM) supplementation. The results were normalized to those of cells infected with scrambled shRNA. GOT1 expression was detected by immunoblotting. f, OCR analysis of Pooled VDAC3 knockout and control HeLa cells. VDAC3 expression was detected by immunoblotting. g, DHOA uptake into proteoliposomes containing VDAC3. Empty liposomes with glycerol were used as a control. h, A schematic indicating how the VDAC3 channel acts as a platform to integrate the pyrimidine biosynthetic complex. (a-g) Values are shown as the mean ± SD, n = 3 (a-e,g) or 4 (f) biologically independent samples. P values were obtained using one-way ANOVA with Tukey test (a), two-way ANOVA with Sidak test (b-d) and unpaired two-tailed Student’s t-test (e,g). (b-e,g) Western blots are verified in three replicates with similar results.
