Fig 1: shMTHFR enhanced cytosolic and nuclear SHMT/DHFR/TYMS protein complex, MTHFD1 (A), as well as DNA repair protein MLH1, and p53 expressions (B) in folate repletion and folate depletion. The quantifications of SHMT/DHFR/TYMS and MTHFD1 are shown in Table 4. The total abundance of the SHMT/DHFR/TYMS protein complex (combined cytosol and nucleus) significantly increased by shMTHFR, especially under folate depletion. Folate depletion induced MTHFD1 expression in shMTHFR and such induction was more drastic in the nucleus in response to folate depletion (Table 4B). These results may account in part for the stable isotopic tracer experiments using L- [3-13C]-serine. After cells were initially depleted of folate and then supplemented with low dose folinate, deoxythymidine monophosphate (dTMP) enrichments (dT+1 represents M+1 of dTMP) from [3-13C]-serine were significantly higher in shMTHFR compared to Neg and WT (Table 5). In addition, shMTHFR induced hMLH1 and p53 expression in both folate depletion and repletion, consisting with the finding of reduced DNA instability (Table 6A,B) in shMTHFR. The quantifications of MLH1 and p53 protein expression are shown in Table 6C. Moreover, folate restriction decreased nuclear and cytosol hMLH1 and p53 protein abundance, and shMTHFR recovered the reduction of hMLH1 and p53, especially in the nucleus (Figure 3B, Table 6C). Abbreviations: SHMT1, serine hydroxymethyl-transferase1; TS, thymidylate synthase; DHFR, dihydrofolate reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase; hMLH1, human Mut L homologue-1.