Transfection of HCT-116 cells with PFKFB3 siRNA inhibits F2,6BP, glucose uptake, ATP and S6K/S6 phosphorylation and simultaneously increases ROS. HCT-116 cells were transfected with either a control siRNA (ctrl) or 10 nM of a siRNA directed against PFKFB3 (PFKFB3). Total protein was harvested 48 hours post-transfection and protein levels relative to β-actin were determined by Western blotting (A). Densitometry data are presented as the mean fold change ± SD from three experiments (B). F2,6BP levels were determined using an enzyme-coupled assay (C). Glucose uptake was estimated based on the uptake of 2DG (D). After 48 hours of transfection with either a control or a PFKFB3-specific siRNA, protein expression of both phosphorylated and total levels of downstream mTOR targets, p70S6K and ribosomal protein S6 (S6) and of AMPK and ULK1 were measured using Western blotting (E). Quantitative densitometry is reported as phosphorylated protein relative to total protein for p70S6K, S6, AMPK and ULK1 (F). ATP was measured using a bioluminescence assay (G) and reactive oxygen species were measured after loading the cells with DCFDA using flow cytometry (H). Data are presented as the mean fold change ± SD of three experiments (*P <0.05).