Fig. 4: SE-FUdR promotes dTMP depletion in E. coli and C. elegans.

Throughout this figure: % hatchlings and LC–MS data were analyzed as described in Fig. 1. Statistical significance was assessed via two-tailed unpaired nonparametric t-test for % hatchlings quantification. LC–MS data were analyzed using one-tailed ratio t-test after ROUT outlier treatment. Data are presented as mean values ± SEM, scale bars = 200 µm. n = # independent biological replicates. Source data are provided as a Source Data file. a Quantification of progeny viability of C. elegans exposed to subLth-FUdR (1 μg/mL) while cultured on EORB1 (RNAi-competent derivative of HB101) carrying empty RNAi empty vector (EV) or RNAi against tyms-1. n = 3. See EORB1 strain development in Methods and Supplementary Fig. 8. b Representative images of progeny viability of C. elegans exposed to subLth-FUdR (0.25 μg/mL) while cultured on WT (BW25113) or thyA KO E. coli lawns. c Quantification of b treatments. n = 3. d Representative images of progeny viability of C. elegans exposed to subLth-FUdR (0.1 μg/mL) + serine 1.5 mg/mL while cultured on WT (BW25113) or thyA KO E. coli lawns. e Quantification of d treatments. n = 3. f Quantification of progeny viability of C. elegans exposed to subLth-FUdR ± serine while cultured on WT (BW25113) or tdk KO E. coli lawns. n = 3. g LC–MS measurement of dTMP normalized to [¹³C9,¹⁵N2]UMP (Norm dTMP) in E. coli WT (BW25113) and lpd KO, and C. elegans cultured in these two E. coli strains. The ratio Norm dTMP in SE-FUdR / Norm dTMP in subLth-FUdR is depicted for each treatment. n = 3. h Setup of dTMP-rescue experiment: (1) upp;udk double KO lawn avoids enhanced-FUMP toxicity otherwise driven by thymidine; (2) 5′-fluoroorotic acid (5-FO) as a source of FUMP; (3) subLth-FUdR + serine to promote SE-FUdR; (4) ±dTMP to test rescue of SE-FUdR toxicity. i Representative images of progeny viability of C. elegans cultured on upp;udk double KO E. coli lawns ±5-FO, ±SE-FUdR (0.25 μg/mL FUdR and 1.5 mg/mL serine), ±dTMP (1.5 μg/mL) showing dTMP rescues SE-FUdR toxicity. j Quantification of treatments in i (denoted with asterisks) and other controls. n = 3. k Working model of SE-FUdR toxicity. Through promoting 5,10-mTHF synthesis, dietary serine enables FdUMP-mediated inhibition of E. coli TS (ThyA). The consequent scarcity of dietary dTMP then exacerbates the toxic effect of sublethal FUdR, leading to DNA toxicity, and death of the worm.