Extended Data Figure 3. FTCD depletion enables cancer cells to maintain THF pools and nucleotide synthesis even when treated with methotrexate (part 2).

a-b. Pool sizes of glycine (a) and serine (b) are not significantly different between FTCD-depleted and control cells. Glycine and serine levels were measured in vehicle- or methotrexate-treated HEL and Ramos cells. p-values were calculated by one-way ANOVA. n=3, biological replicates. c, d. Greater cellular pool of 5,10-methenyl THF in methotrexate-treated cells following FTCD depletion. [U-¹³C] serine labeling of 5,10-methenyl THF showed higher abundance of unlabeled 5,10-methenyl THF in FTCD-depleted cells, compared to control cells. This implies higher availability of 5,10-methenyl THF in these cells at the time of labeling onset, which agrees with the higher levels of both THF and 5,10-methenyl THF in the methotrexate-treated FTCD-depleted cells compared to the methotrexate-treated control cells. c. 5,10-methenyl THF levels were measured by LC/MS in vehicle- and methotrexate-treated HEL and Ramos cells. 5,10-methenyl THF levels were normalized to aminopterin as an internal standard. p-values were calculated by one-way ANOVA. n=3 biological replicates. d. HEL cells were treated with 5 μM methotrexate for 48 hours, and Ramos cells were treated with 20 μM methotrexate for 72 hours to decrease THF levels and nucleotide synthesis in WT cells. The media was then replaced with [U-¹³C] serine-containing media, and cells were incubated in [U-¹³C] serine plus methotrexate for additional 24 hours followed by cell harvesting and LC/MS analysis. Higher abundance of unlabeled 5,10-methenyl THF implies higher availability of reduced folate at the time of labeling onset. 5,10-methenyl THF levels were normalized to aminopterine as an internal standard. p-values were calculated for the unlabeled fraction by one-way ANOVA. n=3, biological replicates.