Glucose production assay

A glucose production assay was performed according to previously published methods (20). In brief, HepG2 cells were seeded in 6-well plates at a density of 5×10⁵ cells per well and exposed to indicated concentrations of ITCZ for 6 h, followed by washing twice with PBS to remove the remaining glucose. The cells were then incubated with 100 µl glucose production medium containing 2 mM of sodium pyruvate and 20 mM of sodium lactate (Sigma-Aldrich; Merck KGaA) for 4 h. 50 µl supernatant was collected and the glucose concentration was measured by a Glucose (HK) Assay kit (Sigma-Aldrich; Merck KGaA) following the manufacturer's instructions.