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. 2023 Jun 9;14:3390. doi: 10.1038/s41467-023-38859-x

Fig. 4. Effect of nutrient supplements on CD15-3-treated cells indicate broad inhibition of folate pathway.

Fig. 4

A Bar plot showing how metabolite supplementation impacts growth rate in the presence of CD15-3. Rescue from CD15-3-induced growth inhibition is observed with thymidine, serine, NAG and IMP supplementations. A fixed concentration of 200 µM CD15-3 was used in these supplementation experiments. n = 3 biologically independent samples were used for the experiments. Data are presented as mean values +/- SEM. Source data are provided as a Source Data file. B A metabolic supplementation strategy was employed for target identification by utilizing metabolic pathway analysis to find supplementation patterns consistent with inhibition of a particular metabolic pathway. Flux balance analysis was used to identify metabolic pathways with inhibition consistent with observed growth rescue patterns. C Purine biosynthesis pathway. Both IMP and AMP supplements are downstream of folate-requiring steps. However, AMP is unable to be converted to dGTP due to an irreversible step in the pathway, while IMP can be converted to both dATP and dGTP. D Pyrimidine biosynthesis pathway. The growth-rescuing supplement, thymidine, enters the pathway downstream of the folate-requiring step. Supplements that enter upstream of the folate-requiring step, namely orotate and uridine, did not rescue growth. E Serine and glycine biosynthesis. Serine, which produces the charged folate form 5,10-methylene tetrahydrofolate when converted to glycine, rescued growth, while glycine itself did not rescue growth. F Correlation of growth rate from different supplements with purine biosynthesis metabolites and corresponding model calculated growth benefit following a constraint on the folate-dependent reaction AICART in purine biosynthesis. G Correlation of growth rate from different supplements with pyrimidine biosynthesis metabolites and corresponding model calculated growth benefit following a constraint on the folate-dependent reaction TMDS in pyrimidine biosynthesis. H Correlation of growth rate from different supplements with serine metabolism metabolites and corresponding model calculated growth benefit in the form of folate cofactor regeneration.