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. Author manuscript; available in PMC: 2022 May 18.
Published in final edited form as: Nat Metab. 2021 Nov 18;3(11):1500–1511. doi: 10.1038/s42255-021-00486-5

Figure 3. MTR is required for nucleotide synthesis in 5-methyl THF medium.

Figure 3.

(A-D) LC/MS measurement of intracellular AMP, ADP, ATP (A), GMP, GDP, GTP (B), GAR, AICAR, IMP (C), and dTMP (D) levels in A549 MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured for 4 days in folic acid (+FA) or 5-methyl THF (5-MeTHF) after a folate prestarvation period (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: AMP: 0.006, ADP: 0.003, ATP: 0.015, GMP: 0.004, GDP: 0.007, GTP: 0.011, GAR: 0.002, AICAR: 0.003, IMP: 0.007, dTMP: 0.79; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: AMP: 0.01, ADP: 0.022, ATP: 0.005, GMP: 0.019, GDP: 0.015, GTP: 0.009, GAR: 0.003, AICAR: 0.005, IMP: 0.025, dTMP: 0.001; +EV +FA vs +EV +5-methyl THF p-values: AMP: 0.0002, ADP: 0.008, ATP: 0.0006, GMP: 0.54, GDP: 0.0004, GTP: 0.0007, GAR: 0.01, AICAR: 0.07, IMP: 0.016, dTMP: 0.019). Data are normalized to protein concentration and an internal standard. Mean +/− SEM error bars are displayed. (E-F) Proliferation rates of A549 MTR knockout cells without +EV or +MTR cultured as in A-D in the indicated folate, with or without the addition of 100 μM each of the indicated nucleotide precursors hypoxanthine (H), uridine (U), thymidine (T) (n = 3 independent samples; except +MTR +H,U,T n = 6) or the addition of 1 mM serine or formate (n = 3 independent samples). Mean +/− SD error bars are displayed. p-values are derived from a two-tailed, unpaired Welch’s t test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).