![Increased incorporation of glucose carbon through glycolysis and the pentose phosphate pathway in mutant JAK2–expressing HSPCs. (A) Schematic of glucose labeling and tracing in BM LSK and MEP cells. Fluorescence-activated cell sorted (FACS) LSK cells and MEPs were cultured with [U-13C6] d-glucose for 8 hours and analyzed by ion chromatography–Fourier transform mass spectrometry (IC-FTMS) to determine the fate of labeled carbon atoms derived from glucose in various downstream metabolites. (B) Bar graphs depicting the levels (μmoles per gram of protein) of glucose-derived 13C containing isotopologues of metabolites in glycolysis and pentose phosphate pathways. LSK and MEP cells were collected and pooled from 9 to 12 mice of matching genotype as 1 replicate. Data are from 2 independent experiments. Insert showing the key rate-limiting step in glycolysis in which the activity of Pfkfb enzyme potentiated activity of rate-limiting enzyme phosphofructokinase (Pfk) through generation of fructose-2,6-biphosphate (F2,6BP) from fructose-6-phosphate (F6P). 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-1 (3PO) is a specific pharmacological inhibitor of Pfkfb3 isoform. (C) Pfkfb1-4 mRNA expression in MEP cells (n = 3 per genotype). (D) Pfkfb3 protein expression in BM LSK and MEP cells as measured by intracellular FACS (n = 3 per genotype). (E) Graphical representation of increased glycolysis rate-limiting step involving conversion of F6P to F1,6BP and its positive regulator PFKFB3, which can be inhibited by 3PO. All data are presented as mean ± standard error of the mean. See also supplemental Figure 6. ***P < .001. 6PG, 6-phosphogluconate; 6PGL, 6-phosphogluconolactone; ADP, adenosine 5′-diphosphate; ATP, adenosine triphosphate; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; G6P, glucose 6-phosphate; PRPP, phosphoribosyl pyrophosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; TCA, tricarboxylic acid cycle; Xu5P, xylulose 5-phosphate.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/134/21/10.1182_blood.2019000162/7/bloodbld2019000162f5.png?Expires=1743236021&Signature=AwgsY~ebggO0mT79qjQNdngirPoYkSI4IB4E-4EUaZw7SmIRsmyLbkD-lXcc4wFhDlJUuWgWb9Yfunwu4HM0367ATvywKKzURNfmBq7xTQBwEBRI5ldKx7uG90maw0oQ29fbXBlh5~nVSIJ-YecufZseHjb4zt68nwz27NKbzNH4OI0k3hAE0a00yAhIVHq-zCpgJIblQ4qDhxY9wiEm0vq9eWWo5JZmR09vUlld7fmzXCOi~lJJa8SBFIMNZiPjXf2gJ6~zjonVyjN9lOmkh4m2w1wAAZM4-Al7S~fAWbuyUKT~IxFZjA6CXJRaYiQwTGa-OzFYR1HI-tzp4sdymA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Increased incorporation of glucose carbon through glycolysis and the pentose phosphate pathway in mutant JAK2–expressing HSPCs. (A) Schematic of glucose labeling and tracing in BM LSK and MEP cells. Fluorescence-activated cell sorted (FACS) LSK cells and MEPs were cultured with [U-13C6] d-glucose for 8 hours and analyzed by ion chromatography–Fourier transform mass spectrometry (IC-FTMS) to determine the fate of labeled carbon atoms derived from glucose in various downstream metabolites. (B) Bar graphs depicting the levels (μmoles per gram of protein) of glucose-derived 13C containing isotopologues of metabolites in glycolysis and pentose phosphate pathways. LSK and MEP cells were collected and pooled from 9 to 12 mice of matching genotype as 1 replicate. Data are from 2 independent experiments. Insert showing the key rate-limiting step in glycolysis in which the activity of Pfkfb enzyme potentiated activity of rate-limiting enzyme phosphofructokinase (Pfk) through generation of fructose-2,6-biphosphate (F2,6BP) from fructose-6-phosphate (F6P). 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-1 (3PO) is a specific pharmacological inhibitor of Pfkfb3 isoform. (C) Pfkfb1-4 mRNA expression in MEP cells (n = 3 per genotype). (D) Pfkfb3 protein expression in BM LSK and MEP cells as measured by intracellular FACS (n = 3 per genotype). (E) Graphical representation of increased glycolysis rate-limiting step involving conversion of F6P to F1,6BP and its positive regulator PFKFB3, which can be inhibited by 3PO. All data are presented as mean ± standard error of the mean. See also supplemental Figure 6. ***P < .001. 6PG, 6-phosphogluconate; 6PGL, 6-phosphogluconolactone; ADP, adenosine 5′-diphosphate; ATP, adenosine triphosphate; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; G6P, glucose 6-phosphate; PRPP, phosphoribosyl pyrophosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; TCA, tricarboxylic acid cycle; Xu5P, xylulose 5-phosphate.