Fig. 2. TPP enables de novo asparagine synthesis and proliferation under ASNase treatment.

(A) Immunoblot analysis of vector control and two clonal TPK1 KOs made from Jurkat cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Fold change in cell number (log₂) of vector control, TPK1_KO1, and TPK1_KO2 Jurkat cells, after untreated or 0.0005 U/ml ASNase conditions for 5 days (mean ± SD, n = 3). Statistics: P < 0.05 by two-tailed unpaired t test for equal variances, for all 15 untreated-treated pairs. (C) Schematic depicting a metabolic route of asparagine synthesis from glutamine. Filled circles represent 13C atoms derived from [U-¹³C]-glutamine. (D to H) Total abundance [a.u. (arbitrary units)] of indicated metabolites derived from labeled glutamine in vector control, TPK1_KO1, and cDNA-rescued TPK1_KO1 Jurkat cells. Cells were incubated for 24 hours in medium containing [U-¹³C]-glutamine (2 mM) in the presence or absence of asparagine (378 μM) and TPP (3 μM). Colors indicate mass isotopologs (mean ± SD, n = 3).