The rate of oxygen consumption was thus measured Selleck Nutlin-3a using a Clark oxygen electrode in three clonal populations of stable VCP KD SH-SY5Y cells. VCP protein expression levels were reduced by approximately 90% in stable VCP KD SH-SY5Y cells compared to stable SCR SH-SY5Y cells ( Figure S1B). The basal rate of oxygen consumption was significantly increased

in VCP KD cells compared to control ( Figure 3A; for numbers see Table S2). Furthermore, addition of oligomycin resulted in an inhibition of oxygen consumption in control cells but not in VCP KD cells, while the uncoupler FCCP increased oxygen consumption to the same maximal values in both cell populations. Calculation of Vbasal/Voligomycin and Vbasal/VFCCP revealed a decrease in the Vbasal/Voligomycin index, suggesting an increase in mitochondrial respiration Rapamycin ( Figure 3B; for numbers see Table S2). No differences in the Vbasal/VFCCP index were observed between VCP KD and SCR cells, showing that ETC complexes are not damaged and they are working at a similar rate under normal conditions ( Figure 3C; for numbers see Table S2). To further analyze ETC and OXPHOS functionality and coupling, we permeabilized stable VCP KD and SCR cell lines using a low concentration of digitonin (40 μM) and basal oxygen consumption rates were measured

in the presence of external substrates for the ETC in the absence of ADP. We then added 50 nmol ADP to establish state 3 (V3) respiration. Upon consumption of this ADP, mitochondria resumed an inhibited state, termed state 4 (V4). Respiratory control ratio (RCR) is the ratio of V3 to V4 and is considered an indicator of coupling of OXPHOS and respiration. RCR values confirmed the uncoupling of mitochondrial respiratory chain from

oxidative phosphorylation in stable VCP KD cells compared to control ( Figure 3D; for numbers see Table S3). The “ADP/O” ratio, expressed as the oxygen consumed per nmol ADP added during V3, indicates the efficiency of oxidative phosphorylation. ADP/O ratios indicated that oxidative phosphorylation efficiency was reduced by more than 30% in VCP KD cells compared to SCR cells ( Figure 3E; for numbers see next Table S3). Taken together, these data show that the respiratory rate, driven by the loss of potential and oxidation of the NADH pool, is increased in VCP-deficient cells and that VCP deficiency increases the mitochondrial proton leak causing uncoupling between respiration and OXPHOS. Mitochondrial uncoupling may occur through a variety of mechanisms including altered lipid peroxidation. Using the fluorescent ratiometric oxidation-sensitive dye C11 BODIPY581/591, we therefore determined the levels of lipid peroxidation in VCP-deficient cells.