Fig. 2|. Shared and unique transcriptional response to the related fluoropyrimidines 5-fluorouracil (5-FU) and capecitabine (CAP) during aerobic and anaerobic growth.

(a) Principal component analysis of E. coli MG1655 transcriptomes under different treatments in aerobic and anaerobic conditions (n=3 biological replicates; 2 independent experiments were conducted for aerobic growth). (b,c) Volcano plots of differentially expressed genes (DEGs) [False Discovery Rate (FDR)<0.1, |log₂ fold-change|>1, significance limits are marked with dash lines; Wald test with Benjamini-Hochberg correction]. DEGs involved in pyrimidine metabolism are labeled by gene name and marked with a red dot. preT and preA are labeled in blue. (d) Pathway enrichment analysis following 5-FU and CAP exposure under aerobic and anaerobic conditions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to test for pathway enrichment (q-value<0.1, Hypergeometric test with Benjamini-Hochberg correction from enrichKEGG function in clusterProfiler⁹⁸). (e) Upset plot comparing the fluoropyrimidine responsive DEGs under different growth conditions (Supplementary Table 5). The numbers on top of the vertical bars represent the number of DEGs unique to a single comparison (single dot) or shared among multiple comparisons (connected dots). The set size refers to a total number of DEGs in a single comparison (DEGs defined as FDR<0.1, |log₂ fold-change|>1; Wald test with Benjamini-Hochberg correction). (f) Heatmap of transcriptional changes of 44 genes involved in flagellar biosynthesis in response to fluoropyrimidine treatments under anaerobic and anaerobic conditions. Gray boxes indicate non-significant changes (FDR>0.1). Bolded genes indicate DEGs that are down-regulated in 5-FU evolved strains reported in literature³⁶. (g) preT and preA transcript levels are significantly lower during anaerobic growth relative to aerobic growth irrespective of the presence of fluoropyrimidines (q-value<0.1, DESeq).