Fig. 2. Thr is catabolized to maintain the SAM/SAH ratio in mESCs.

(A) Schematic of Thr-SAM pathway activity in pluripotent stem cells, relative to MEFs. (B) HPLC analysis of ¹⁴C-labeled amino acids derived from [U-¹⁴C]Thr in mESCs after 24h. Scintillation counts per minute (CPM) are plotted against retention time. (C) Fraction of intracellular metabolites derived from [U-¹³C]Thr in mESCs over 5h, as measured by SRM analysis (n=3). (D) Steady-state fraction of intracellular metabolites derived from [U-¹³C]Thr, [U-¹³C]Ser, [U-¹³C]-glucose, or [U-¹³C]Gln in mESCs after 48h, as measured by SRM analysis (n=3). (E) SRM analysis of metabolite abundances in mESCs during 6h of Thr restriction, relative to time zero (n=3). (F) SRM analysis of several metabolic ratios over a 6h time course, relative to time zero (n=3). (G) Feeder-free mESCs were subjected to Thr restriction for 12h, then supplemented for 36h with the indicated metabolites at the given concentrations. Alkaline phosphatase-positive colonies were quantified and normalized to mESC colony numbers in normal mESC media (% colonies recovered). X denotes relative concentration with respect to DMEM medium. NAC, N-acetyl-cysteine. Py, pyruvate. DMG, dimethylglycine. Bet, betaine. H, hypoxanthine. T, thymidine. All error bars represent the s.e.m. from three independent measurements.