Measurement of Glucose Uptake Activity on HepG2 Cells

Glucose uptake activity assay of rh-aFGF₁₃₅ in vitro was performed essentially as described below. HepG2 cells were seeded in 96-well plates at a density of 6 × 10³ cells per well (100 μL of medium per well; 6 wells per treatment condition) and incubated overnight at 37°C in a 5% CO₂ humidified incubator. After overnight starvation, the cells were stimulated with 3.7, 11.1, 33.3, 100, 300, and 900 ng/mL rh-aFGF₁₃₅ for 24 h. Glucose uptake was measured with a glucose assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol. The absorbance at 510 nm was recorded using a microplate reader (Molecular Devices, Shanghai, China), and the glucose consumption rate was calculated.