Extended Data Fig. 2. MTR knockout affects levels of intracellular folate species and proliferation in different folate sources.

(A) Proliferation rates of A549 MTR knockout cells without (+EV) or with MTR expression (+MTR) cultured in folic acid (+FA) or 96% 5-methyl THF, 4% folic acid (phys ratio)⁴ for 4 days after a folate pre-starvation period (n = 3 independent samples). (B) LC/MS measurement of intracellular folate, 5-methyl THF, combined 5,10-methenyl THF/10-formyl THF, and 5-formyl THF levels in A549 and T.T MTR knockout cells +EV or +MTR cultured up to 3 days in medium lacking folates (n = 1 independent sample for each time point). Data are normalized to protein concentration and an internal standard. (C) Proliferation rate of A549 or T.T MTR knockout cells +EV or +MTR cultured in medium lacking folates (prestarved) or with folic acid for 3 days (n = 3 independent samples; A549: p = 0.011; T.T: p = 0.046). p values indicated are derived from a two-tailed, unpaired Welch’s t test (* = p < 0.05). (A,C) Mean +/− SD error bars are displayed.