Fig. 5. VEGF induces PFKP expression, PFK enzyme activity, aerobic glycolysis, and proliferation in GBM cells.

WB and qRT-PCR were performed with indicated primers and antibodies, respectively (D, F–I). A, B Serum-starved GSCs were treated with VEGF (20 ng/mL). Glucose consumption (A) and lactate secretion (B) were analyzed. C GSCs in 0.1% serum medium were treated with VEGF (20 ng/mL). WST-8 assay was then performed. D, E Serum-starved GSCs were treated with VEGF (20 ng/mL). Indicated protein expression levels (D) and PFK enzymatic activity (E) were measured. F Serum-starved GSCs were pretreated with DMSO or MK-2206 (5 μM) for 1 h and then stimulated with VEGF (20 ng/mL) for 30 min. G Serum-starved GSCs were pretreated with VEGF (20 ng/mL) for 1 h and then treated with cycloheximide (CHX;100 μg/mL) in the presence of DMSO or MK-2206 (5 μM). Quantification of PFKP levels relative to tubulin is shown (bottom panel). H Serum-starved GSCs were pretreated with DMSO or MK-2206 (5 μM) for 1 h and then stimulated with or without VEGF (20 ng/mL) for 24 h. I Serum-starved GSCs were pretreated with DMSO or SU1498 (30 μM) for 1 h and then stimulated with or without VEGF (20 ng/mL) for 24 h. J–L LN229 cells with or without expression of PFKP shRNA and with or without the reconstituted expression of WT Flag-rPFKP or Flag-rPFKP S386A were cultured in serum-free DMEM with or without VEGF (20 ng/mL) for 24 h. PFK enzymatic activity (J), glucose consumption (K), and lactate secretion (L) were then analyzed. M LN229 cells with or without the expression of PFKP shRNA and with or without the reconstituted expression of WT Flag-rPFKP or Flag-rPFKP S386A were cultured in 0.1% serum medium with or without VEGF (20 ng/mL). WST-8 assay was then performed. Data are presented as mean ± SD of three independent experiments (A–C, E, G, J–M). *P < 0.05; **P < 0.01; ***P < 0.001, based on the Student’s t-test or one-way ANOVA with Tukey’s post hoc test.