DiscussionHepG2 cells, like all cancer cells, generate energy from glucose by glycolysis, with little contribution from oxidative phosphorylation (OXPHOS). However, when glucose is replaced by galactose in the growth media, these cells use OXPHOS to generate energy by utilizing glutamine and asparagine in a process called anapleurosis. Comparison of cell viability in the different media is now used routinely to screen drugs for mitochondrial toxicity. Here using a novel In-Cell Proteomics method, we have expanded the evaluation of the effect of the classical inhibitors of OXPHOS: FCCP (uncoupler), oligomycin (ATP synthase inhibitor), myxathiazol (complex III inhibitor), and rotenone (Complex I inhibitor) on mitochondrial function and cell metabolism. Cells were incubated with compounds at 100uM in either glucose or galactose for 6, 24 or 72h prior to fixation. Protein levels were quantified using specific antibodies. All 4 compounds induced cell death by 6h in galactose based on cell appearance and reduction in MCl1 and PUMA levels. In parallel, there was dephosphorylation of both 4E-BP1 and the S6 ribosomal protein, indicating mTor involvement, and a strongly increased phosphorylation of eIF2α, consistent with a component of oxidative stress. In glucose there was no cell death at 6h with FCCP or oligomycin, but some apoptosis with rotenone and myxathiazol. Unlike galactose all 4 compounds induced a large increase in phosphorylation of both p38MAPK and c-Jun kinases in glucose along with increases in both fatty acid oxidation and synthesis along with ER stress based on the increased levels of ACADM, HADHB, ACC2 and CHOP1 respectively. Our results indicate considerable effect of OXPHOS inhibitors outside mitochondria, some but not all of which can be accounted for by loss of ATP production, indicating that other factors such as ROS and altered metabolic intermediates exiting this organelle provide retrograde signalling of mitochondrial stress to the rest of the cell.