Fig. 1.

Validation of methyl donor depletion in UD-SCC2 cells. Disruption to the one carbon metabolism cycle leads to an increase in extracellular homocysteine and loss of intracellular methionine, choline, and betaine. Extracellular homocysteine concentration was measured by HPLC and normalised to cell number. a Concentration of extracellular homocysteine increased in UD-SCC2 cells following 72 and 168 h culture in depleted (5%) methyl donor medium compared to control cells (100%). The intracellular levels of b methionine, c betaine, and d choline, measured by LC-MS/MS were all significantly decreased in UD-SCC2 cells upon methyl donor depletion compared to controls (*p < 0.05) and showed signs of recovery after repletion in 100% methyl donor culture conditions for 72 h. n = 3 independent experiments performed in triplicate; one-way independent ANOVA with a Bonferroni post-hoc comparison