Polarstar omega system

Cell proliferation was measured by MTT assays and cell counting analysis. For the MTT assay, cells were plated in 96-well plates and cultured for 72 h; then, 10 μL of 10 mg/mL MTT stock was added to each well and incubated for another 4 h. After dissolving the cells in dimethyl sulfoxide (DMSO), the absorbance was read with a POLARstar Omega system (BMG Labtech, VIC, Australia). For the cell growth curves generated by cell counting analysis, cells were seeded in six-well plates, and cell numbers were counted every other day after trypan blue staining.
For the colony formation assay, cells were seeded in 60-mm dishes and cultured for 10 days. The colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet in 20% methanol for 15 min, then washed with PBS and photographed.

PFK1 activity was determined as described previously [51 (link)]. Briefly, the reaction was performed using cell lysates (10 µg) in 1 mL of reaction buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 5 mM Na₂HPO₄, 1 mM NH₄Cl, 0.1 mM AMP, 1 mM ATP, 0.2 mM NADH, 5 mM fructose-6-phosphate, 5 U of triosephosphate isomerase, 1 U of α-glycerophosphate dehydrogenase (Sigma-Aldrich) and 1 U of aldolase (Aladdin, Shanghai, China). Absorbance was recorded at 340 nm at room temperature every 15 s for 10 min using a POLARstar Omega system (BMG Labtech). One unit of PFK1 activity was defined as the amount of enzyme that catalyzes the conversion of 1 µM fructose-6-phosphate to fructose-1,6-bisphosphate per min, and the data are presented as relative PFK1 activity levels.
The PK activity of the cell lysates (4 µg) was measured with a PK assay kit (BioVision, Mounysin View, CA, USA) according to the manufacturer’s instructions.