Glutamine supports both mitochondrial function and glutathione production following FLT3 TK inhibition. (A) Total and isotopologue levels of TCA cycle intermediates, glutamate and reduced glutathione (GSH), measured by LC-MS analysis in MV411 cells treated with AC220 1 nM or vehicle control and grown in media containing U-¹³C₅ and ¹⁵nitrogen [¹⁵N₂] glutamine (GLN) (mean ± SEM, n = 5; ***P = .0005, **P = .0017, *P = .0195 for citrate, P = .0228 for malate, P = .0216 for glutamate; ns, not significant; 2-tailed, paired Student t test). A schematic representation of glutamine metabolism and labeling pattern of metabolites is also provided. (B) Percentage of total levels of TCA cycle metabolites labeled by U-¹³C₅,¹⁵N₂-GLN through its oxidative metabolism measured by LC-MS analysis in MV411 cells treated with AC220 1 nM or vehicle control (mean ± SEM, n = 5; ****P < .0001, ***P = .0008, **P = .0067; 2-way ANOVA with Bonferroni’s multiple comparisons). (C) Relative mitochondrial membrane potential of MV411 cells treated with AC220 1 nM or vehicle control (left) with representative flow cytometry histogram (right) (mean ± SEM, n = 3; * P = .0145; 2-tailed, paired Student t test). (D) Relative mitochondrial mass of MV411 cells treated with AC220 1 nM or vehicle control (left) with representative flow cytometry histogram (right panel) (mean ± SEM, n = 3; ns, not significant; P = .13; 2-tailed paired t test). (E) Relative GSH/GSSG ratio, GSH, and GSSG of MV411 cells treated with AC220 1 nM or vehicle control (mean ± SEM, n = 6; * P = .0173; ns, not significant; 2-tailed, paired Student t test). (F) Percentage of total levels of glutamine, glutamate, GSH, and GSSG labeled by U-¹³C₅,¹⁵N₂-GLN measured by LC-MS analysis in MV411 cells treated as in panel A (mean ± SEM, n = 5; **** P < .0001; ns, not significant; 2-way ANOVA with Bonferroni’s multiple comparisons. (G) Relative GSH levels of MV411 cells treated with AC220 1 nM or vehicle control in the presence or absence of glutamine (mean ± SEM, n = 5; **P = .0017, *P = .0471, q = 4.248; ANOVA with Tukey’s multiple comparisons). (H) Relative cytoplasmic ROS levels of MV411 cells treated with AC220 1 nM or vehicle control in the presence or absence of glutamine (mean ± SEM, n = 14; ****P < .0001; ANOVA with Tukey’s multiple comparisons).