Extended Data Figure 3. Effects of glycine cleavage system inhibition on cells with high or low SHMT2 expression levels.

a, Overview of the serine hydroxymethyltransferase and glycine cleavage reactions mediated by SHMT2 and GLDC, respectively. Asterisks indicate metabolites that are labeled with ¹³C during mitochondrial metabolism of U-¹³C serine. Only upon completion of both reactions will ¹³C-labeled CO₂ be formed, which is captured and detected by scintillation as described in Methods. b, Measurement of ¹³CO₂ production, a readout of sequential SHMT2 and GLDC activity on [U-¹³C]-serine, in intact mitochondria isolated from LN229s expressing shGFP or shSHMT2_1, as described in Methods. c, Table indicating cell viability in various cell lines following transduction of GLDC shRNAs for 6–7 days. Values are relative to the CTG signal of the same cells secondarily transduced with shGFP and grown in parallel. d, SHMT2 and GLDC expression in LN229 cells stably transduced with shSHMT2_1 or shGFP, then secondarily transduced shGLDC or shGFP as indicated. e, Viability of LN229 cells first transduced with control or SHMT2 shRNAs, then transduced with shGFP or GLDC shRNAs for 5 days. Values are relative to that of cells secondarily transduced with shGFP. f, Viability of U251 cells first transduced with control or SHMT2 shRNAs, then transduced with shGFP or GLDC shRNAs for 7 days. Values are relative to that of cells secondarily transduced with shGFP. g,h,i, Viability of various cell lines transduced with shRNAs targeting (g) GLDC, (h) GCSH (glycine cleavage system protein H, another integral component of the glycine cleavage system), or (i) SHMT2. Values are relative to those of the cells expressing shGFP, which were grown in parallel. For b,c, and e–i, n=3 independent biological replicates; error bars are SD. *P<0.05 (student’s t test).