Penicillin streptomycin antibiotic

In accordance with the approved protocol (IAEC/17-I/05), the hepatocytes were harvested from adult Swiss albino mice (25–30 g) as per previously reported method.25 (link) The viability of cells was determined by the following formula: Cells/mL = (Total viable cells in 4 squares)/4 x dilution factor x 10⁴.
The cells were diluted and seeded into collagen pre-coated T75 culture flasks at a density of 1.75×10⁵ cells mL⁻¹. The harvested murine hepatocytes were aseptically cultured in sterile Dulbecco’s Modified Eagle Medium (DMEM; HiMedia Laboratories, India), supplemented with FBS (12%) and Penicillin-Streptomycin Antibiotic (1%, HiMedia Laboratories, India) at 37°C under humidified CO₂ (5%) (Shel lab, USA).
Upon reaching 70–90% confluency, the hepatocytes were trypsinized (Trypsin, 0.25% + EDTA, 0.02%), centrifuged (800 rpm x 8 min), re-suspended, counted, and seeded (1x10⁵ cells/2 mL well) on collagen pre-coated well-plate. For next 48 h, the cells were grown either in glucose-free DMEM (DMEM - glucose), DMEM + glucose (4.5 g mL⁻¹), DMEM- glucose + fructose (0.55 mM), DMEM - glucose + fructose (1 mM) or DMEM - glucose + fructose (1 mM) + Insulin (0.1 µM), and grouped as NC, Glu, FC1, FC2 and FC3, respectively.

The human hepatocellular carcinoma cell line (HepG2) was sourced from National Centre for Cell Sciences (NCCS, Pune, India) and grown under standard aseptic conditions using sterile Dulbecco’s Modified Eagle Medium (DMEM; HiMedia Laboratories, India), supplemented with FBS (12%) and Penicillin-Streptomycin Antibiotic (1%, HiMedia Laboratories, India) at 37°C under humidified CO₂ (5%) (Shel lab, USA). The cells were seeded aseptically (1x10⁵ cells/2mL well) and allowed to grow for 48 h, either in DMEM (NC), DMEM + 0.55 mM fructose (FC1), DMEM + 1mM fructose (FC2) or DMEM + 1 mM fructose + 0.1 µM Insulin (FC3). The HepG2 cells (FC1-FC3) were exposed to either PG-HM, its fractions (PG-H, PG-C, PG-EA, PG-B, PG-A at 35 µg mL⁻¹); or DMSO (0.1% v/v, VC1–VC3). The supernatant and cell lysates were collected and preserved at −80°C for further analysis of glycogen, carbohydrate metabolizing enzymes (hexokinase, aldehyde dehydrogenase, ketohexokinase, phosphofructokinase), secondary messenger of insulin signaling (PI3K p-tyr-STAT-3, mTOR), hypoxia and inflammation (HIF-1α, VEGF, TNF-α), using commercially available kits in accordance with the manufacturer’s instructions.