Oxytherm clark type electrode system

Oxygen consumption was measured using an Oxytherm Clark-type electrode system (Hansatech, Norfolk, UK) as described previously with modifications^{32 (link)}. HepG2 cells (2 × 10⁷) were incubated with 2 µM LW1564 for 6 h and harvested, and then the OCR was measured for 5 min at 37 °C with a thermos-regulated circulating system. For mechanistic studies, digitonin-permeabilized HepG2 cells (2 × 10⁷) were added to a detection device containing 2 ml of respiration buffer (0.25 M sucrose, 2 mM KH₂PO₄, 5 mM MgCl₂, 1 mM EDTA, 1 mM ADP, 20 mM MOPS, pH 7.4). Then, the following substrates, which provide electrons to components within the ETC, were added: 5 mM sodium pyruvate, 5 mM sodium malate, 5 mM sodium succinate, 5 mM L-ascorbic acid and 0.2 mM N,N,N,N-tetramethyl-p-phenylenediamine (TMPD). Complex I, III and IV inhibitors (rotenone, antimycin A and KCN, respectively) were added to final concentrations of 1 µM.