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. 2016 Sep;57(9):1428–1435. doi: 10.2967/jnumed.115.167387

FIGURE 2.

FIGURE 2.

PFKFB3 mediates increase in 2-deoxyglucose uptake in human macrophages treated with GM-CSF. (A) Intracellular levels of Fru-2,6-P2 were determined at the indicated times with and without GM-CSF and in absence or presence of PFKFB3 inhibitor 3PO (5 μM). GM-CSF augments Fru-2,6-P2 levels, which are attenuated in presence of 3PO, demonstrating critical role of expression of PFKFB3 after GM-CSF treatment. (B) Lactate accumulation in culture medium was determined at 18 h with and without native or heat-inactivated GM-CSF and in absence or presence of 3PO (5 μM). Lactate levels (by-product of glycolysis) increase with GM-CSF exposure and are dampened by 3PO. (C) TNF-α levels were determined in culture medium after challenge with native or heat-inactivated GM-CSF. (D) Intracellular levels of Fru-2,6-P2 were determined at the indicated times with GM-CSF and in presence of neutralizing anti-TNF-α antibody. GM-CSF augments Fru-2,6-P2 levels, which are attenuated in presence of neutralizing anti-TNF-α antibody. (E) 2-deoxyglucose uptake was determined at the indicated times with and without GM-CSF and in absence or presence of neutralizing anti-TNF-α antibody (20 ng/mL) or PFKFB3 inhibitor 3PO (5 μM). 2-deoxyglucose significantly increases with addition of GM-CSF. This effect was diminished with neutralizing anti-TNF-α antibody and with 3PO. (F) To analyze contribution of PFKFB3 to enhancement of glycolytic flux elicited by GM-CSF, macrophages were transfected for 18 h with specific siRNA to silence PFKFB3 or with corresponding inactive RNA control (scRNA) and in absence or presence of 3PO, followed by activation for 18 h with GM-CSF. 2-deoxyglucose uptake was significantly decreased after silencing PFKFB3 compared with corresponding control (scRNA) or inhibition with 3PO. Data are mean ± SD. *P < 0.05 vs. same condition in control (absence of GM-CSF or 0 h). **P < 0.01 vs. same condition in control (absence of GM-CSF or 0 h). #P < 0.05 for GM-CSF vs. heated GM-CSF, with 3PO plus GM-CSF. ##P < 0.01 for GM-CSF vs. heated GM-CSF, with 3PO plus GM-CSF. IgG vs. anti-TNF-α antibody or scRNA vs. siPFKFB3 with or without 3PO. scRNA = scrambled RNA; siRNA = silencer RNA.