Fig. 4: BCAT transamination supplies nitrogen for aspartate and nucleotide biosynthesis in ccRCC.

a Diagram of the labeling pattern originating from ¹⁵N leucine catabolism. The gray circles indicate unlabeled N, blue circle ¹⁵N while white circles represent unlabeled carbons. Measured metabolites through LC–MS are indicated in blue circles. BCAT1/2 branched-chain amino acid transaminase 1/2, GOT1/2 glutamic-oxaloacetic transaminase 1/2, ASNS asparagine synthase, ASS1 argininosuccinate synthase, ASL argininosuccinate lyase. Abundance of labeled leucine m+1 and glutamate m+1 (b), aspartate m+1 and asparagine m+1 (c) originating from ¹⁵N leucine after 27 h normalized to total ion count. Data represent the mean of 6 independent cultures ± SD. p-values were calculated using one-way ANOVA where each group was compared with HK2. d Proportion of total pool of the indicated labeled metabolites originating from ¹⁵N leucine after 24 h in culture with EBSS + FBS 2.5% for 24 h. Data are normalized to total ion count and represent the mean of 6 independent cultures ± SD. e–g Intracellular abundance of the indicated metabolites after treatment with BCATI 100 μM in Plasmax for 22 h. Values are normalized to total ion count and expressed as the mean of 3 independent cultures ± SD. p-values were calculated using one-way ANOVA with multiple comparisons and indicated in the graph for the comparisons treated vs. vehicle for all biological groups. h Proliferation rate of the indicated cell lines in the presence of 100 μM of BCATI in Plasmax. Data represent the mean of 3 independent experiments ± S.E.M. Values represent fold-change increase of growth relative to day 0.