F2,6P2 and glucose uptake measurements

Intracellular F2,6P₂ concentration was determined following the method as previously described [18]. Briefly, after treatment with dedicated concentration of PFK15 for 12 h, cell extracts were incubated in the mixture contained 50 mmol/L Tris/HCl, 5 mmol/L MgC1₂, 0.15 mmol/L NADH, 1 mmol/L F6P (fructose 6-phosphate), 10 units/liter PPi (pyrophosphate)-dependent PFK1, 0.45 kilounit/liter aldolase, 5 kilounit/liter triose-phosphate isomerase, and 1.7 kilounit/liter glycerol-3-phoshate dehydrogenase (Sigma). Then, absorbance (OD = 339 nm) changes were analyzed after 0.5 mmol/L pyrophosphate was added. The F2,6P₂ content was finally calculated and normalized to total cellular protein.

Glucose uptake was determined using the Glucose Assay Kit (Sigma) following the manufacturer’s instructions. Briefly, MKN45 cells were plated in complete medium and treated with PFK15 (0–10 μmol/L) for 12 h. Cells were then washed and starved in serum-free medium for 12 h, followed by 2-Deoxyglucose incubation for 1 h. After treatment, cells were lysed and the absorbance was measured at 412 nm.