Fig. 4

Glutamine usage is interlinked with asparagine synthase (ASNS) in ADPKD. a Schematic overview of the conversion of glutamine into glutamate by transamidating aspartate into asparagine by ASNS. b Labelled ¹⁵N-asparagine is significantly increased in the Pkd1 mutant MEFs, c total levels of asparagine are increased compared to the control cells and d decreasing levels of aspartate (M + 0), expressed as percentage relative to controls. e Isotopologue distribution of intracellular asparagine showing that the pool coming from glutamine (¹⁵N₁ and ¹⁵N₂) is significantly decreased in siAsns compared to the mock Pkd1^−/− and control cells. f Total α-KG was significantly decreased in siAsns compared to the mock Pkd1^−/− and control cells in the ¹⁵N₂ glutamine labelling. g Total asparagine was significantly decreased in siAsns compared to the mock Pkd1^−/− cells. h α-KG (M + 5) in the ¹³C₅-glutamine labelling was significantly decreased in siAsns compared to the mock Pkd1^−/− and control cells. Data are means from six technical replicates from one experiment. i Representative graph showing the percentage of cell count compared to the respective mock controls. Silencing Asns, deprivation of glucose or both treatments in Pkd1^+/+ resulted in no significant difference, whilsts each condition resulted in a significant reduction in cell number with an additive effect of Asns silencing with glucose starvation in Pkd1^−/− cells. Data are means from three technical replicates from two independent experiments. Mean ± SEM were indicated, n.s. not significant (P ≥ 0.05), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, t-test for b, c, and e. ANOVA followed by Bonferroni for e, f, g, h, and i