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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 4. MTR knockout reduces SAM but not methionine levels.

Figure 4.

(A) Proliferation rates of A549 MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured in folic acid (+FA) or 5-methyl THF (5-MeTHF) or in 100 uM each nucleotide precursor (hypoxanthine, uridine, and thymidine) for 4 days after a folate prestarvation period, at varied extracellular methionine concentrations (n = 3 independent samples, except n = 6 for +FA 100 uM methionine +EV/+MTR). (B-C) LC/MS measurement of intracellular methionine, serine, lysine (B), SAM, and SAH levels (C) in A549 MTR knockout cells +EV or +MTR cultured in the indicated folate as in A (n = 3 independent samples; +EV +FA vs +MTR +FA p-values: methionine: 0.01, serine: 0.0001, lysine: 0.936, SAM: 0.0009, SAH: 0.012; +EV +5-methyl THF vs +MTR +5-methyl THF p-values: methionine: 0.003, serine: 0.007, lysine: 0.0001, SAM: 0.012, SAH: 0.152; +EV +FA vs +EV +5-methyl THF p-values: methionine: 0.001, serine: 0.01, lysine: 9.1*10−6, SAM: 0.47, SAH: 0.1). Data are normalized to protein concentration and an internal standard. (D) Western blots to assess phosphorylation of mTORC1 targets and levels of mono- or dimethyl lysine-containing proteins in A549 MTR knockout cells +EV or +MTR cultured in the indicated folate for 16 hours. The ratio of phospho-protein to total protein signal from 3 independent replicates is shown. (E) Proliferation rates of A549 MTR knockout cells +EV or +MTR cultured in the indicated folate as in A, with or without the addition of 1 mM SAM (n = 3 independent samples for +EV, n = 6 for +MTR; +EV −folate vs +EV +SAM p = 0.084, +EV +SAM vs +MTR +SAM p = 0.001). (A,E) Mean +/− SD error bars are displayed. (B-D) Mean +/− SEM 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).