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. Author manuscript; available in PMC: 2018 Jul 26.
Published in final edited form as: Blood. 2018 Feb 20;131(15):1639–1653. doi: 10.1182/blood-2017-12-820035

Figure 3. Glutamine supports both mitochondrial function and glutathione production following FLT3 tyrosine kinase inhibition.

Figure 3

(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 ± s.e.m., n=5, *** P=0.0005, ** P=0.0017, * P=0.0195 for citrate, P=0.0228 for malate, P=0.0216 for glutamate, ns, not significant, two-tailed paired t-test). A schematic representation of glutamine metabolism and labelling pattern of metabolites by is also provided. (B) Percentage of total levels of TCA cycle metabolites labelled 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 ± s.e.m., n=5, **** P<0.0001, *** P=0.0008, ** P=0.0067, two-way ANOVA with Bonferroni’s multiple comparisons). (C) Relative mitochondrial membrane potential of MV411 cells treated with AC220 1 nM or vehicle control (left panel) with representative flow-cytometry histogram (right panel) (mean ± s.e.m., n=3, * P=0.0145, two-tailed paired t-test). (D) Relative mitochondrial mass of MV411 cells treated with AC220 1 nM or vehicle control (left panel) with representative flow-cytometry histogram (right panel) (mean ± s.e.m., n=3, ns, not significant, P=0.13, two-tailed paired t-test). (E) Relative GSH/GSSG ratio, GSH and GSSG of MV411 cells treated with AC220 1 nM or vehicle control (mean ± s.e.m., n=6, * P=0.0173, ns, not significant, two-tailed paired t-test). (F) Percentage of total levels of glutamine, glutamate, GSH and GSSG labelled by U-13C5,15N2-GLN measured by LC-MS analysis in MV411 cells treated as in (A) (mean ± s.e.m., n=5, **** P<0.0001, ns, not significant, two-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 ± s.e.m., n=5, ** P=0.0017, * P=0.0471, q=4.248, DF=12, 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 ± s.e.m., n=14, **** P<0.0001, ANOVA with Tukey’s multiple comparisons).