The metabolic effect of SHMT2 inhibition. (A) Metabolites significantly regulated upon inducible SHMT2 knockdown (shSHMT2#2, d3 upon doxycyline induction) from LC/MS analysis (n = 6 each). The heatmap is color coded by row-wise z-scores of metabolite abundances. AICAR, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside; GSH, glutathione; IMP, inosine monophosphate. (B) Metabolite abundance for selected significantly regulated metabolites from LC/MS analysis (up to n = 6 each; median is shown). (C) Cell viability assay (MTT assay) for BL60 and SU-DHL4 after 48 hours of SHIN1 treatment and supplementation with indicated metabolites. (0.1 mM glycine and 0.3 mM serine correspond to regular medium concentration.) Nucleoside components are described in the supplemental Methods. Data were normalized to medium control (n = 3-4; mean ± SEM is shown). Glycine supplementation: [BL60] P = .0052, [SU-DHL4] ns (two-way ANOVA), Glycine/formate supplementation: [BL60] P = .0032, [SU-DHL4] ns (two-way ANOVA). Bonferroni posttest for glycine supplementation at 3.3 mM: [BL60] 1 µM SHIN1 P < .01; 5 and 10 µM SHIN1 P < .001; [SU-DHL4] 10 µM SHIN1 P < .001; for glycine/formate supplementation at 3.3/2 mM: [BL60] 1 µM SHIN1 P < .01; 5 to 10 µM SHIN1 P < .001; [SU-DHL4] 5 µM SHIN1 P < .05, 10 µM SHIN1 P < .001.