Poly d lysine coated xfe24 plates

For real-time measurement of glycolytic rates, the Seahorse Bioscience XFe24 extracellular flux analyzer (CA, USA) was used according to manufacturer's protocol. Hepa1-6 cells were seeded in Poly-d-lysine coated XFe24 plates (Gibco, Montana, USA) at 2500 cells/well and kept at 37 °C and 5 % CO2 overnight. Cells were transfected with either scramble or H19 siRNA (5 nM, 48h). On termination of incubation, cells were washed with XF base medium DMEM without phenol red containing 2 mM glutamine, and pH was set to 7.4 ± 0.2. Concentration of inhibitors used were 10 mM glucose, 2 μM oligomycin, 100 mM 2-Deoxy-d-glucose. All experiments were performed in triplicate and normalized by total protein.

The Seahorse Bioscience XFe24 extracellular flux analyzer (Agilent, CA, USA) was used to quantify the real-time mitochondrial oxygen consumption rate (OCR) according to manufacturer's protocol. Hepa1-6 cells were seeded in Poly-d-lysine coated XFe24 plates (Gibco, Montana, USA) at 2500 cells/well and kept at 37 °C and 5 % CO₂ overnight. Cells were transfected with either scramble or H19 siRNA (5 nM, 48h) alone or with VDAC siRNA (5 nM) or with the empty vector or VDAC1 clone (1 μg). On termination of incubation, cells were washed three times with XF base medium DMEM without phenol red containing 2 mM glutamine, 1 mM sodium pyruvate and 10 mM of glucose and pH was set to 7.4 ± 0.2. Concentration of inhibitors used were 2 μM oligomycin, 1.25 μM FCCP, 2 μM of rotenone and antimycinA. All experiments were performed in triplicate and normalized to the total protein.