Fig 1: V-ATPase inhibition alters cellular metabolism. HUH7 cells were treated as indicated (24 h). a Cells were exposed sequentially to oligomycin, FCCP and rotenone/antimycin. Vertical lines indicate time of addition of mitochondrial inhibitors. Oxygen consumption rate (OCR) was measured over time using a Seahorse XFe96 Analyzer. Cell mito stress test was performed according to manufacturer’s protocol. b Respiratory parameters were calculated from OCR data (a) according to mitochondrial stress test protocol (User Guide Kit 103015–100 Agilent). c Mitochondrial fuel flex test was performed according to manufacturer’s protocol (User Manual Kit 103270–100 Agilent). Fuel dependency was calculated as described in the manual. d Cells were loaded with MitoSOX™ and mitochondrial superoxide (SOX) was quantified by flow cytometry. e NADPH and NADP+ levels were assessed using NADP/NADPH-Glo™ luminescence-based assay as described by the manufacturer (G9081 Promega). NADPH to NADP+ ratio was calculated and normalized to DMSO control. f Cytosolic cytochrome C was detected by flow cytometry. Bars are the mean + SEM of three independent experiments. p* < 0.05 (One-way ANOVA, Dunnett post test) (g) Protein levels of active Caspase 3 and Parp-1 cleavage have been determined and quantified by Western Blot. h Cartoon of mode of action. V-ATPase inhibition leads to transcriptional regulation of PGC1α and PPAR α. Lipid droplets (LD) are changed in size and localization, leading to cardiolipin depletion, fission and impairment of mitochondrial function. This results in induction of mitochondria-driven apoptosis