Figure 1 from Mitochondrial Transfer Rescues Respiration to Support De Novo Pyrimidine Biosynthesis and Tumor Progression

DHODH-dependent respiration delays acquisition of mtDNA in ρ0 cells. A, 4T1 ρ0 and 4T1 ρ0 AOX cells were subcutaneously injected into Balb/c mice at 106 per animal. On days 3, 5, 10, 15, 20, and 25, tumor tissue was retrieved from the mice, and sublines were established. Parental cells and sublines derived from 4T1 ρ0 and 4T1 ρ0 AOX cells were assessed for mtDNA using qPCR. B, Parental, ρ0 D15, and 4T1 ρ0 AOX D25 cells were assessed for the distribution of mtDNA polymorphism in tRNAArg, D-loop, and 16S rRNA in single cells of lines. C, Heatmap illustrates mRNA expression of mitochondrial and nuclear-encoded OXPHOS genes in the cell lines indicated. The patterns were generated by the median of clustering analysis of bulk RNA sequencing data. The vertical axis represents individual samples, whereas the horizontal axis shows individual transcripts. D, Protein expression of AOX was assessed in parental cells, and sublines were derived from ρ0 AOX. E and F, Routine and DHODH-dependent respiration of parental, ρ0, ρ0 AOX, and the derived sublines was evaluated using the Oxygraph. G and H, DHODH activity was assessed in parental and ρ0, ρ0 D3, ρ0 D15, and ρ0 AOX sublines and NAD+/NADH ratio for parental ρ0, ρ0 AOX. I, Parental and ρ0, ρ0 D3, ρ0 D5, ρ0 D15, ρ0 AOX, ρ0 AOX D15, ρ0 AOX D20, and ρ0 AOX D25 cells were evaluated by native blue gel electrophoresis for the assembly of respiratory complexes and supercomplexes using antibodies to relevant subunits as shown. J, Balb/c mice were grafted subcutaneously with parental, ρ0, and ρ0 AOX cells (106 per animal), and tumor volume was assessed on days as indicated using calipers (n = 3 for each group). Data are derived from at least three independent experiments. Statistical analysis was performed using ordinary one-way ANOVA and the GraphPad Prism 8 software, considering differences with the P value of ≤0.05 as statistically significant. *, P value of 0.01 to 0.05; **, P value of 0.001 to 0.01; ***, P value of 0.0001 to 0.001; ****, P < 0.0001.